Antibodies and assays for detection of folate receptor 1

ABSTRACT

The invention generally relates to antibodies that bind to human folate receptor and diagnostic assays for folate receptor 1-based therapies. Methods of using the antibodies to monitor therapy are further provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 15/473,008, filed Mar. 29, 2017, which is a divisional applicationof U.S. application Ser. No. 14/473,828, filed Aug. 29, 2014, whichclaims the priority benefit of U.S. Provisional Application Nos.61/872,407, filed Aug. 30, 2013; U.S. 61/875,475, filed Sep. 9, 2013;and U.S. 61/940,184, filed Feb. 14, 2014, each of which is herebyincorporated by reference herein in its entirety.

REFERENCE TO A SEQUENCE LISTING SUBMITTED ELECTRONICALLY VIA EFS-WEB

The Sequence Listing submitted Jun. 7, 2018, as a text file named“2921.0440005_SequenceListing_ST25,” created on Jun. 6, 2018, and havinga size of 68,602 bytes, is hereby incorporated by reference pursuant to37 C.F.R. § 1.52(e)5).

FIELD OF THE INVENTION

The field of this invention generally relates to diagnostic assays andkits for folate receptor 1-based therapies and antibodies that bind tohuman folate receptor 1.

BACKGROUND OF THE INVENTION

Cancer is one of the leading causes of death in the developed world,with over one million people diagnosed with cancer and 500,000 deathsper year in the United States alone. Overall it is estimated that morethan 1 in 3 people will develop some form of cancer during theirlifetime. There are more than 200 different types of cancer, four ofwhich-breast, lung, colorectal, and prostate—account for over half ofall new cases (Jemal et al., 2003, Cancer J. Clin. 53:5-26).

Folate Receptor 1 (FOLR1), also known as Folate Receptor-alpha or FolateBinding Protein, is an N-glycoslated protein expressed on plasmamembrane of cells. FOLR1 has a high affinity for folic acid and forseveral reduced folic acid derivatives. FOLR1 mediates delivery of thephysiological folate, 5-methyltetrahydrofolate, to the interior ofcells.

FOLR1 is overexpressed in the vast majority of ovarian cancers, as wellas in many uterine, endometrial, pancreatic, renal, lung, and breastcancers, while the expression of FOLR1 on normal tissues is restrictedto the apical membrane of epithelial cells in the kidney proximaltubules, alveolar pneumocytes of the lung, bladder, testes, choroidplexus, and thyroid (Weitman S D, et al., Cancer Res 52; 3396-3401(1992); Antony A C, Annu Rev Nutr 16: 501-521 (1996); Kalli K R. et al.Gynecol Oncol 108: 619-626 (2008)). This expression pattern of FOLR1makes it a desirable target for FOLR1-directed cancer therapy.

Because ovarian cancer is typically asymptomatic until advanced stage,it is often diagnosed at a late stage and has poor prognosis whentreated with currently available procedures, typically chemotherapeuticdrugs after surgical de-bulking (von Gruenigen V et al., Cancer 112:2221-2227 (2008); Ayhan A et al., Am J Obstet Gynecol 196: 81 e81-86(2007); Harry V N et al., Obstet Gynecol Surv 64; 548-560 (2009)). Thusthere is a clear unmet medical need for more effective diagnostics forovarian cancers.

Some previous assays used to detect shed FOLR1 are not sufficientlyspecific to FOLR1. For example, some assays do not distinguish betweenFOLR1 and other folate receptor family members (FOLR2, 3, & 4) or reportvalues for total FBP (Folate Binding Protein). Additionally, some assaysrequire that human samples (e.g., plasma) be pre-treated with a lightacid wash step to dissociate folic acid from the receptor. Some assayresults may also have inaccuracies due to competitive effects betweenthe antibody therapy and diagnostic antibody. Additionally, manycommercially available kits are traditionally unreliable both in theirreagents, and in their lot-to-lot stability. Evaluations of these kitshave given very mixed results, and are intended for research use only.Many require that the human sample be pre-diluted before analysis toreduce the chance of false positives due to the “matrix effect.” Thus,there is a clear need for highly sensitive and accurate diagnosticassays that can detect a clinically relevant dynamic range of FOLR1 as acompanion for FOLR1-based therapies.

SUMMARY OF THE INVENTION

Anti-FOLR1 antibodies and antigen-binding fragments thereof as well asmethods for detecting FOLR1, diagnosing FOLR1-mediated diseases anddisorders (such as cancer), monitoring the efficacy of anti-FOLR1therapies, optimizing anti-FOLR1 therapies, and stratifying patients areall provided herein.

The anti-FOLR1 antibodies provided herein can have a diagnostic role.For example, the anti-FOLR1 antibodies provided herein to distinguishbetween tumor and non-tumor cells or tissues or to identify tumor types,subtypes, or grades. In one embodiment, an anti-FOLR1 antibody providedherein and/or a FOLR1-detection assay provided herein can be used todistinguish between subtypes of non-small cell lung cancer (NSCLC)including adenocarcinoma and squamous cell carcinoma as describedherein. In another embodiment, an anti-FOLR1 antibody provided hereinand/or a FOLR1-detection assay provided herein can be used to rule out atype of cancer (e.g., to determine that a cell or tissue is not a typeof cancer), for example, sarcoma.

In some embodiments, an antibody or antigen-binding fragment thereofprovided herein can specifically bind to an epitope of FOLR1, whereinthe epitope comprises at least one, at least two, or three N-glycoslatedamino acids. Glycosylation can be critical for membrane localization.See e.g., Yan et al., J. Am. Soc. Nephol. 13: 1385-1389 (2002).Advantageously, the antibodies and antigen-binding fragments herein candetect FOLR1 expression on cell membranes and detect a clinicallyrelevant dynamic range of FOLR1. The more discreet staining obtainedwith the antibodies and antigen-binding fragments provided herein allowsfor discrimination among samples all grouped together as high expressionlevels (with a score of 3) using antibodies that bind to different FOLR1epitopes, lack sufficient specificity, and/or lack sufficientsensitivity.

In some embodiments, an antibody or antigen-binding fragment thereofprovided herein can specifically bind to the same FOLR1 epitope as anantibody selected from the group consisting of: (a) an antibodycomprising the polypeptide of SEQ ID NO:27 and the polypeptide of SEQ IDNO:28; (b) an antibody comprising the polypeptide of SEQ ID NO:29 andthe polypeptide of SEQ ID NO:30; (c) an antibody comprising thepolypeptide of SEQ ID NO:31 and the polypeptide of SEQ ID NO:32; (d) anantibody comprising the polypeptide of SEQ ID NO:62 and the polypeptideof SEQ ID NO:63 or SEQ ID NO:64; and (e) an antibody comprising thepolypeptide of SEQ ID NO:65 and the polypeptide of SEQ ID NO:66 or SEQID NO:67. In some embodiments, the epitope comprises an N-glycosylatedamino acid.

In some embodiments, an antibody or antigen-binding fragment thereofprovided herein can specifically bind to FOLR1, wherein said antibody orfragment thereof competitively inhibits binding to FOLR1 of an antibodyselected from the group consisting of: (a) an antibody comprising thepolypeptide of SEQ ID NO:27 and the polypeptide of SEQ ID NO:28; (b) anantibody comprising the polypeptide of SEQ ID NO:29 and the polypeptideof SEQ ID NO:30; (c) an antibody comprising the polypeptide of SEQ IDNO:31 and the polypeptide of SEQ ID NO:32; (d) an antibody comprisingthe polypeptide of SEQ ID NO:62 and the polypeptide of SEQ ID NO:63 orSEQ ID NO:64; and (e) an antibody comprising the polypeptide of SEQ IDNO:65 and the polypeptide of SEQ ID NO:66 or SEQ ID NO:67.

In some embodiments, the antibody or antigen-binding fragment thereofcomprises the VH CDR1-3 and VL CDR1-3 polypeptide sequences selectedfrom the group consisting of: (a) SEQ ID NOs:3-8, respectively; (b) SEQID NOs:9-14, respectively; (c) SEQ ID NOs:15-20, respectively; (d) SEQID NOs:21-26, respectively; (e) SEQ ID NOs: 3-5 and SEQ ID NOs: 59, 7,and 8, respectively; (f) SEQ ID NOs: 3, 60, and 5 and SEQ ID NOs: 6-8,respectively; (g) SEQ ID NOs: 3, 61, and 5 and SEQ ID NOs: 6-8,respectively; (h) SEQ ID NOs: 3, 60, and 5 and SEQ ID NOs: 59, 7, and 8,respectively; and (i) SEQ ID NOs: 3, 61, and 5 and SEQ ID NOs: 59, 7,and 8, respectively.

In some embodiments, an antibody or antigen-binding fragment thereofprovided herein can specifically bind to FOLR1, wherein the antibody orfragment thereof comprises the VH CDR1-3 and VL CDR1-3 polypeptidesequences selected from the group consisting of: (a) SEQ ID NOs:3-8,respectively; (b) SEQ ID NOs:9-14, respectively; (c) SEQ ID NOs:15-20,respectively; (d) SEQ ID NOs:21-26, respectively; (e) SEQ ID NOs: 3-5and SEQ ID NOs: 59, 7, and 8, respectively; (f) SEQ ID NOs: 3, 60, and 5and SEQ ID NOs: 6-8, respectively; (g) SEQ ID NOs: 3, 61, and 5 and SEQID NOs: 6-8, respectively; (h) SEQ ID NOs: 3, 60, and 5 and SEQ ID NOs:59, 7, and 8, respectively; (i) SEQ ID NOs: 3, 61, and 5 and SEQ ID NOs:59, 7, and 8, respectively; and (j) variants of (a) to (i) comprising 1,2, 3, or 4 conservative amino acid substitutions.

In some embodiments, the antibody or antigen-binding fragment thereofcomprises polypeptide sequences that are at least 90%, at least 95%, orat lesast 99% identical to polypeptide sequences selected from the groupconsisting of: (a) SEQ ID NO:27 and SEQ ID NO:28; (b) SEQ ID NO:29 andSEQ ID NO:30; (c) SEQ ID NO:31 and SEQ ID NO:32; (d) SEQ ID NO:62 andSEQ ID NO:63 or SEQ ID NO:64; (e) SEQ ID NO:65 and SEQ ID NO:66 or SEQID NO:67; (f) SEQ ID NO:68 and SEQ ID NO:69. In some embodiments, thepolypeptide sequences comprise, consist essentially of, or consist ofthe amino acids of sequences selected from the group consisting of: (a)SEQ ID NO:27 and SEQ ID NO:28; (b) SEQ ID NO:29 and SEQ ID NO:30; (c)SEQ ID NO:31 and SEQ ID NO:32; (d) SEQ ID NO:62 and SEQ ID NO:63 or SEQID NO:64; (e) SEQ ID NO:65 and SEQ ID NO:66 or SEQ ID NO:67; (f) SEQ IDNO:68 and SEQ ID NO:69.

In some embodiments, an antibody or antigen-binding fragment thereofprovided herein can specifically bind to FOLR1, wherein the antibody orfragment thereof comprises a humanized heavy chain variable regioncomprising CDR1, CDR2, and CDR3 regions comprising the amino acids ofSEQ ID NO:51, SEQ ID NO:52 or 53, and SEQ ID NO:54, respectively, ahumanized light chain variable region comprising CDR1, CDR2, and CDR3regions comprising the amino acids of SEQ ID NO:48, SEQ ID NO:49, andSEQ ID NO:50, respectively, and a murine constant region. In someembodiments, the humanized heavy chain variable region comprises theamino acids of SEQ ID NO:45 and the humanized light chain variableregion comprises the amino acids of SEQ ID NO:47.

In some embodiments, the antibody or antigen-binding fragment thereof isrecombinantly produced. In some embodiments, the antibody orantigen-binding fragment thereof is murine, non-human, humanized,chimeric, resurfaced, or human. In some embodiments, the antibody orantigen-binding fragment thereof binds to human FOLR1 but not FOLR2 orFOLR3. In some embodiments, the antibody or antigen-binding fragmentthereof is a full length antibody. In some embodiments, the antibody orantigen-binding fragment thereof is an antigen-binding fragment. In someembodiments, the antibody or antigen-binding fragment thereof comprises,consists essentiall of, or consist of a Fab, Fab′, F(ab′)2, Fd, singlechain Fv or scFv, disulfide linked Fv, V-NAR domain, IgNar, intrabody,IgGΔCH2, minibody, F(ab′)3, tetrabody, triabody, diabody, single-domainantibody, DVD-Ig, Fcab, mAb2, (scFv)2, or scFv-Fc.

In some embodiments, a polypeptide provided herein can specifically bindFOLR1, wherein the polypeptide comprises sequences selected from thegroup consisting of: (a) SEQ ID NOs:3-8, respectively; (b) SEQ IDNOs:9-14, respectively; (c) SEQ ID NOs:15-20, respectively; (d) SEQ IDNOs:21-26, respectively; (e) SEQ ID NOs: 3-5 and SEQ ID NOs: 59, 7, and8, respectively; (f) SEQ ID NOs: 3, 60, and 5 and SEQ ID NOs: 6-8,respectively; (g) SEQ ID NOs: 3, 61, and 5 and SEQ ID NOs: 6-8,respectively (h) SEQ ID NOs: 3, 60, and 5 and SEQ ID NOs: 59, 7, and 8,respectively; (i) SEQ ID NOs: 3, 61, and 5 and SEQ ID NOs: 59, 7, and 8,respectively; and (j) variants of (a) to (i) comprising 1, 2, 3, or 4conservative amino acid substitutions. In some embodiments, thepolypeptide comprises sequences that are at least 90%, at least 95%, orat least 99% identical to sequences selected from the group consistingof: (a) SEQ ID NO:27 and SEQ ID NO:28; (b) SEQ ID NO:29 and SEQ IDNO:30; (c) SEQ ID NO:31 and SEQ ID NO:32; (d) SEQ ID NO:62 and SEQ IDNO:63 or SEQ ID NO:64; (e) SEQ ID NO:65 and SEQ ID NO:66 or SEQ IDNO:67; and (f) SEQ ID NO:68 and SEQ ID NO:69. In some embodiments, thepolypeptide comprises the amino acids of (a) SEQ ID NO:27 and SEQ IDNO:28; (b) SEQ ID NO:29 and SEQ ID NO:30; (c) SEQ ID NO:31 and SEQ IDNO:32; (d) SEQ ID NO:62 and SEQ ID NO:63 or SEQ ID NO:64; (e) SEQ IDNO:65 and SEQ ID NO:66 or SEQ ID NO:67; or (f) SEQ ID NO:68 and SEQ IDNO:69.

In some embodiments, the antibody, antigen-binding fragment thereof, orpolypeptide binds to FOLR1 with a Kd of about 0.5 to about 10 nM. Insome embodiments, the antibody, antigen-binding fragment thereof, orpolypeptide binds to a human FOLR1 with a Kd of about 1.0 nM or better.In some embodiments, the binding affinity is measured by flow cytometry,Biacore, ELISA, or radioimmunoassay.

In some embodiments, the antibody, antigen-binding fragment thereof, orpolypeptide binds to an epitope of FOLR1 comprising an amino acid thatis N-glycosylated.

In some embodiments, the antibody, antigen-binding fragment thereof, orpolypeptide is detectably labeled.

In some embodiments, a cell provided herein produces the antibody,antigen-binding fragment thereof, or polypeptide. In some embodiments,the cell is isolated.

Methods of making the antibody, antigen-binding fragment thereof, orpolypeptide are also provided. The methods can comprise (a) culturing acell provided herein; and (b) isolating the antibody, antigen-bindingfragment thereof, or polypeptide from the cultured cell.

Compositions comprising the antibody, antigen-binding fragment thereof,or polypeptide are also provided. In some embodiments, the compositioncomprises the the antibody, antigen-binding fragment thereof, orpolypeptide and buffer selected from the group consisting of: a FACSbuffer, an IHC buffer, and an ELISA buffer.

Methods of using the antibody, antigen-binding fragment thereof, orpolypeptide are also provided.

In some embodiments, a method of detecting FOLR1 expression in a samplecomprises contacting the sample with an antibody, antigen-bindingfragment thereof, polypeptide, or composition provided herein. In someembodiments, the antibody or antigen-binding fragment thereof isdetectably labeled. In some embodiments, the label is selected from thegroup consisting of immunofluorescent label, chemiluminescent label,phosphorescent label, enzyme label, radiolabel, avidin/biotin, colloidalgold particles, colored particles and magnetic particles. In someembodiments, the FOLR1 expression is determined by radioimmunoassay.Western blot assay, cytometry, immunofluorescent assay, enzymeimmunoassay, immunoprecipitation assay, chemiluminescent assay, orimmunohistochemical assay. In some embodiments, the cytometry is flowcytometry. In some embodiments, the FOLR1 expression is determined byIHC.

In some embodiments, a method for increasing the efficacy of cancertherapy with an active agent comprising an anti-FOLR1 antibody orantigen-binding fragment thereof, comprises administering the activeagent to a subject having cancer, wherein increased expression of FOLR1has been detected in a cancerous sample from the subject using anantibody, antigen-binding fragment thereof, polypeptide or compositionprovided herein.

In some embodiments, a method for identifying a cancer likely to respondto an active agent comprising an anti-FOLR1 antibody or antigen-bindingfragment thereof comprises: (a) contacting a biological samplecomprising cells from the cancer with the antibody, antigen-bindingfragment thereof, polypeptide or composition provided herein; (b)detecting binding of the antibody, antibody-fragment, or polypeptide toFOLR1 in the biological sample of (a); (c) assigning a score to thebinding of step (b), wherein the score is assigned based on comparisonto one or more reference samples; and (d) comparing the score in step(c) to the score of a reference tissue or cell, wherein a score for thecancer FOLR1 level that is greater than the score for a normal or lowFOLR1 expressing reference sample or a score for the cancer FOLR1 levelthat is equal to or greater than the score for a high FOLR1 expressingreference sample identifies the cancer as likely to respond to ananti-FOLR1 antibody.

In some embodiments, a method of treating a patient having cancercomprises: (a) determining a FOLR1 expression score from a detection ofFOLR1 expression in a cancerous sample obtained from the patient,wherein the detection is performed using an antibody, antigen-bindingfragment thereof, polypeptide or composition provided herein; and (b)administering an active agent comprising an anti-FOLR1 antibody orantigen-binding fragment thereof to the patient if the score indicatesthe patient will benefit from administration of the active agent.

In some embodiments, a method of treating a patient having cancercomprises (a) determining a FOLR1 expression score from a detection ofFOLR1 expression in a cancerous sample obtained from the patient,wherein the detection is performed using an antibody, antigen-bindingfragment thereof, polypeptide or composition provided herein; and (b)instructing a healthcare provider to administer an active agentcomprising an anti-FOLR1 antibody or antigen-binding fragment thereof tothe patient if the score indicates the patient will benefit fromadministration of the active agent.

In some embodiments, a method of treating a patient having cancercomprises: (a) submitting a cancerous sample taken from a patient havingcancer for determining a FOLR1 expression score from a detection ofFOLR1 expression using an antibody, antigen-binding fragment thereof,polypeptide, or composition provided herein; and (b) administering anactive agent comprising an anti-FOLR1 antibody or antigen-bindingfragment thereof to the patient if the score indicates the patient willbenefit from administration of the active agent.

In some embodiments, a method of treating a patient having cancercomprises: (a) detecting FOLR1 expression in a cancerous sample obtainedfrom the patient, wherein the detection is performed using an antibody,antigen-binding fragment thereof, polypeptide, or composition providedherein; (b) determining a FOLR1 expression score for the canceroussample; and (c) administering an active agent comprising an anti-FOLR1antibody or antigen-binding fragment thereof to the patient if the scoreindicates the patient will benefit from administration of the activeagent.

In some embodiments, a method of treating a patient having cancercomprises: (a) administering to the patient a fixed dose of an activeagent comprising an anti-FOLR1 antibody or antigen-binding fragmentthereof; (b) detecting the patient's FOLR1 relative to the FOLR1 levelin a reference sample, wherein the detection is performed using anantibody, antigen-binding fragment thereof, polypeptide, or compositionprovided herein; and (c) increasing the amount or frequency ofsubsequent fixed doses if the patient's FOLR1 level is elevated.

In some embodiments, a method of optimizing a therapeutic regimen withan active agent comprising an anti-FOLR1 antibody or antigen-bindingfragment thereof for a subject having cancer comprises: (a)administering an increased dose of an active agent comprising ananti-FOLR1 antibody or antigen-binding fragment thereof to a subjecthaving cancer wherein an increased expression of FOLR1 in the subjecthas been detected using an antibody, antigen-binding fragment thereof,polypeptide, or composition provided herein; or (b) administering adecreased dose of the active agent to a subject having cancer wherein adecreased expression of FOLR1 in the subject has been detected.

In some embodiments, a method of optimizing a therapeutic regimen withan active agent comprising an anti-FOLR1 antibody or antigen-bindingfragment thereof for a subject having cancer comprises: (a) detectingthe level of FOLR1 expression in a cancerous sample from the subjectusing an antibody, antigen-binding fragment thereof, polypeptide, orcomposition provided herein; (b) determining a FOLR1 expression scorefor the cancerous sample; and (c) administering an increased dose of anactive agent comprising an anti-FOLR1 antibody or antigen-bindingfragment thereof to the subject if the score is low or administering adecreased dose of the active agent to the subject if the score is high.

In some embodiments, a method of decreasing FOLR1-expressing cancercells in a cancer patient comprises: (a) detecting the FOLR1 level in acancerous sample taken from a patient, compared to the FOLR1 level in areference sample using an antibody, antigen-binding fragment thereof,polypeptide, or composition provided herein; and (b) administering tothe patient a fixed dose of an active agent comprising an anti-FOLR1antibody or antigen-binding fragment thereof if the patient's FOLR1level is elevated compared to the reference sample; wherein theadministration of the active agent decreases the number ofFOLR1-expressing cancer cells in the patient. In some embodiments, amethod of treating cancer in a patient comprises: (a) detecting theFOLR1 level in a cancerous sample taken from a patient, compared to theFOLR1 level in a reference sample using an antibody, antigen-bindingfragment thereof, polypeptide, or composition provided herein; and (b)administering to the patient a fixed dose of an active agent comprisingan anti-FOLR1 antibody or antigen-binding fragment thereof if thepatient's FOLR1 level is elevated compared to the reference sample;wherein the administration of the active agent decreases the size of aFOLR1-expressing tumor or decreases CA125 levels.

In some embodiments, a method of decreasing FOLR1-expressing cancercells in a cancer patient comprises: (a) administering to a patienthaving a cancer a fixed dose of an active agent comprising an anti-FOLR1antibody or antigen-binding fragment thereof: (b) detecting thepatient's FOLR1 level relative to the FOLR1 level in a reference sampleusing an antibody, antigen-binding fragment thereof, polypeptide, orcomposition provided herein; and (c) increasing the amount or frequencyof subsequent fixed doses if the patient's FOLR1 level is elevatedcompared to the reference sample; wherein the administration of theactive agent decreases the number of FOLR1-expressing cancer cells inthe patient. In some embodiments, a method of treating cancer in apatient comprises: (a) administering to a patient having a cancer afixed dose of an active agent comprising an anti-FOLR1 antibody orantigen-binding fragment thereof; (b) detecting the patient's FOLR1level relative to the FOLR1 level in a reference sample using anantibody, antigen-binding fragment thereof, polypeptide, or compositionprovided herein; and (c) increasing the amount or frequency ofsubsequent fixed doses if the patient's FOLR1 level is elevated comparedto the reference sample; wherein the administration of the active agentdecreases the size of a FOLR1-expressing tumor or decreases CA125levels.

In some embodiments, a method of monitoring therapeutic efficacy of afixed dose of an active agent comprising an anti-FOLR1 antibody orantigen-binding fragment thereof in a patient comprises: (a) detecting afirst FOLR1 level in a biological sample from a patient having cancerusing an antibody, antigen-binding fragment thereof, polypeptide, orcomposition provided herein; (b) administering to the patient a fixeddose of an active agent comprising an anti-FOLR1 antibody orantigen-binding fragment; (c) detecting a second FOLR1 level in abiological sample from the patient following active agentadministration, wherein the detecting is performed using an antibody,antigen-binding fragment thereof, polypeptide, or composition providedherein; and (d) comparing the second FOLR1 level to the first FOLR1level; wherein a decrease between the first and second FOLR1 levelindicates therapeutic efficacy.

In some embodiments, a method of identifying a subject having a canceras likely to respond to a low dose anti-FOLR1 treatment regimen,comprises: (a) contacting a biological sample comprising cells from thecancer with an antibody, antigen-binding fragment thereof, polypeptide,or composition provided herein; (b) detecting binding of the antibody,antigen-binding fragment, or polypeptide to the biological sample of(a); (c) assigning a score to the binding of step (b), wherein the scoreis assigned based on comparison to one or more reference samples; and(d) comparing the score in step (c) to the score of a reference tissueor cell, wherein a score for the cancer FOLR1 level that is greater thanthe score for a normal or low FOLR1 expressing reference sample or ascore for the cancer FOLR1 level that is equal to or greater than thescore for a high FOLR1 expressing reference sample identifies the canceras likely to respond to a low dose anti-FOLR1 treatment.

In some embodiments, a method of identifying a cancer as sensitive totreatment with an anti-FOLR1 active agent, comprises: (a) detecting thelevel of FOLR1 expression in a cancerous sample from the cancer using anantibody, antigen-binding fragment thereof, polypeptide, or compositionprovided herein, wherein the detecting comprises the use of a methodthat distinguishes between staining intensity or staining uniformity ina FOLR1 expressing cancerous sample as compared to staining intensity orstaining uniformity in one or more reference samples; (b) determining aFOLR1 staining intensity or staining uniformity score for the canceroussample; and (c) comparing the FOLR1 staining intensity or staininguniformity score determined in step (b) to a relative value determinedby measuring FOLR1 protein expression in at least one reference sample,wherein the at least one reference sample is a tissue, cell, or cellpellet sample which is not sensitive to treatment with an active agentcomprising an anti-FOLR1 antibody or antigen-binding fragment thereofand wherein a FOLR1 staining intensity score for the cancerous sampledetermined in step (b) that is higher than the relative value identifiesthe cancer as being sensitive to treatment with the active agent.

In some embodiments, a method of identifying a cancer as sensitive totreatment with an anti-FOLR1 active agent, comprises: (a) detecting thelevel of FOLR1 expression in a cancerous sample from the cancer using anantibody, antigen-binding fragment thereof, polypeptide, or compositionprovided herein, wherein the detecting comprises the use of a methodthat specifically stains membrane FOLR1 in a FOLR1 expressing canceroussample as compared to membrane FOLR1 in one or more reference samples;(b) determining a FOLR1 score for the cancerous sample; and (c)comparing the FOLR1 score determined in step (b) to a relative valuedetermined by measuring FOLR1 in at least one reference sample, whereinthe at least one reference sample is a tissue, cell, or cell pelletsample which is not sensitive to treatment with an active agentcomprising an anti-FOLR1 antibody or antigen-binding fragment thereofand wherein a FOLR1 score for the cancerous sample determined in step(b) that is higher than the relative value identifies the cancer asbeing sensitive to treatment with the active agent.

In some embodiments, a method of identifying a cancer as sensitive totreatment with an anti-FOLR1 active agent, comprises: (a) detecting thelevel of FOLR1 expression in a cancerous sample from the cancer using anantibody, antigen-binding fragment thereof, polypeptide, or compositionprovided herein, wherein the detecting comprises the use of a methodthat distinguishes between staining intensity or staining uniformity ina FOLR1 expressing cancerous sample as compared to staining intensity orstaining uniformity in one or more reference samples; (b) determining aFOLR1 staining intensity or staining uniformity score for the canceroussample; and (c) comparing the FOLR1 staining intensity or staininguniformity score determined in step (b) to a relative value determinedby measuring FOLR1 protein expression in at least one reference sample,wherein the at least one reference sample is a tissue, cell, or cellpellet sample which is sensitive to treatment with an active agentcomprising an anti-FOLR1 antibody or antigen-binding fragment thereofand wherein a FOLR1 staining intensity score for the cancerous sampledetermined in step (b) that is greater than or equal to the relativevalue identifies the cancer as being sensitive to treatment with theactive agent.

In some embodiments, a method of identifying a cancer as sensitive totreatment with an anti-FOLR1 active agent, comprises: (a) detecting thelevel of FOLR1 expression in a cancerous sample from the cancer using anantibody, antigen-binding fragment thereof, polypeptide, or compositionprovided herein, wherein the detecting comprises the use of a methodthat specifically stains membrane FOLR1 in a FOLR1 expressing canceroussample as compared to membrane FOLR1 in one or more reference samples;(b) determining a FOLR1 score for the cancerous sample; and (c)comparing the FOLR1 score determined in step (b) to a relative valuedetermined by measuring FOLR1 in at least one reference sample, whereinthe at least one reference sample is a tissue, cell, or cell pelletsample which is sensitive to treatment with an active agent comprisingan anti-FOLR1 antibody or antigen-binding fragment thereof and wherein aFOLR1 score for the cancerous sample determined in step (b) that isgreater than or equal to the relative value identifies the cancer asbeing sensitive to treatment with the active agent.

In some embodiments, the method further comprises administering anactive agent comprising an anti-FOLR1 antibody or antigen-bindingfragment thereof to the subject from whom the cancerous sample orbiological sample was obtained.

In some embodiments, the patient's FOLR1 level is detected in acancerous sample or biological sample obtained from the patient. In someembodiments, the cancerous sample or biological sample is a bodilyfluid, cell, or tissue sample. In some embodiments, the cell is acirculating tumor cell. In some embodiments, the bodily fluid is blood,ascites, urine, plasma, serum, or peripheral blood.

In some embodiments, the FOLR1 is membrane localized FOLR1.

In some embodiments, the FOLR1 is shed FOLR1.

In some embodiments, the detecting is by enzyme linked immunosorbentassay (ELISA).

In some embodiments, the detecting is by immunohistochemistry (IHC). Insome embodiments, the IHC is calibrated IHC that can distinguishdifferent levels of FOLR1 expression. In some embodiments, the IHCproduces a range of staining intensity for samples having low cellsurface FOLR1 expression, intermediate FOLR1 cell surface expression, orhigh FOLR1 cell surface expression. In some embodiments, the IHCdistinguishes between staining intensity and staining uniformity in aFOLR1 expressing cancerous sample or biological sample as compared to areference sample. In some embodiments, the IHC detects membrane FOLR1.In some embodiments, the IHC is performed manually. In some embodiments,the IHC is performed using an automated system.

In some embodiments, a FOLR1 score is determined from the IHC.

In some embodiments, a score of at least 1 indicates an increasedexpression of FOLR1 and identifies the cancer as likely to respond to anactive agent comprising an anti-FOLR1 antibody or antigen-bindingfragment thereof.

In some embodiments, a score of at least 2, at least 2 homo (>75%uniformity), or at least 2 hetero (25-75% uniformity) identifies thecancer as likely to respond to an active agent comprising an anti-FOLR1antibody or antigen-binding fragment thereof. In some embodiments, thecancer is lung cancer or endometrial cancer. In some embodiments, ascore of at least 3, at least 3 homo (>75% uniformity), or at least 3hetero (25-75% uniformity) identifies the cancer as likely to respond toan active agent comprising an anti-FOLR1 antibody or antigen-bindingfragment thereof. In some embodiments, the cancer is lung cancer,endometrial cancer, or ovarian cancer.

In some embodiments, an H-score of at least 50 identifies a cancer aslikely to respond to an active agent comprising an anti-FOLR1 antibodyor antigen-binding fragment thereof. In some embodiments, an H-score ofat least 75 identifies an ovarian cancer as likely to respond to anactive agent comprising an anti-FOLR1 antibody or antigen-bindingfragment thereof. In some embodiments, an H-score of at least 50identifies an NSCLC as likely to respond to an active agent comprisingan anti-FOLR1 antibody or antigen-binding fragment thereof. In someembodiments, an H-score of at least 50 identifies an endometrial canceras likely to respond to an active agent comprising na anti-FOLR1antibody or antigen-binding fragment thereof. In one embodiment, anH-score is determined using the FOLR1-2.1 antibody.

In some embodiments, at least 25% of FOLR1 membrane expression in anovarian tumor sample with an intensity of at least 3 identifies theovarian cancer as likely to respond to an active agent comprising ananti-FOLR1 antibody or antigen-binding fragment thereof. In someembodiments, at least 25% of FOLR1 membrane expression in an NSCLCsample with an intensity of at least 2 identifies the NSCLC as likely torespond to an active agent comprising an anti-FOLR1 antibody orantigen-binding fragment thereof. In some embodiments, at least 25% ofFOLR1 membrane expression in an endometrial tumor sample with anintensity of at least 2 identifies the endometrial cancer as likely torespond to an active agent comprising an anti-FOLR1 antibody orantigen-binding fragment thereof. In one embodiment, the expressionscore is determined using the FOLR1-2.1 antibody.

In some embodiments, a score of at least 1 indicates an increasedexpression of FOLR1 and that the patient will benefit fromadministration of an active agent comprising an anti-FOLR1 antibody orantigen-binding fragment thereof. In some embodiments, a score of atleast 2, at least 2 homo (>75% uniformity), or at least 2 hetero (25-75%uniformity) indicates that the patient will benefit from administrationof an active agent comprising an anti-FOLR1 antibody or antigen-bindingfragment thereof. In some embodiments, the cancer is lung cancer orendometrial cancer. In some embodiments, a score of at least 3, at least3 homo (>75% uniformity), or at least 3 hetero (25-75% uniformity)indicates that the patient will benefit from administration of an activeagent comprising an anti-FOLR1 antibody or antigen-binding fragmentthereof. In some embodiments, the cancer is lung cancer, endometrialcancer, or ovarian cancer.

In some embodiments, an H-score of at least 50 indicates that thepatient will benefit from administration of an active agent comprisingan anti-FOLR1 antibody or antigen-binding fragment thereof. In someembodiments, an H-score of at least 75 indicates that a patient withovarian cancer will benefit from administration of an active agentcomprising an anti-FOLR1 antibody or antigen-binding fragment thereof.In some embodiments, an H-score of at least 50 indicates that a patientwith NSCLC will benefit from administration of an active agentcomprising an anti-FOLR1 antibody or antigen-binding fragment thereof.In some embodiments, an H-score of at least 50 indicates that a patientwith endometrial cancer will benefit from administration of an activeagent comprising an anti-FOLR1 antibody or antigen-binding fragmentthereof. In one embodiment, an H-score is determined using the FOLR1-2.1antibody.

In some embodiments, at least 25% of FOLR1 membrane expression in aovarian tumor sample with an intensity of at least 3 indicates that thepatient will benefit from administration of an active agent comprisingan anti-FOLR1 antibody or antigen-binding fragment thereof. In someembodiments, at least 25% of FOLR1 membrane expression in an NSCLCsample with an intensity of at least 2 indicates that the patient willbenefit from administration of an active agent comprising an anti-FOLR1antibody or antigen-binding fragment thereof. In some embodiments, atleast 25% of FOLR1 membrane expression in an endometrial tumor samplewith an intensity of at least 2 indicates that the patient will benefitfrom administration of an active agent comprising an anti-FOLR1 antibodyor antigen-binding fragment thereof. In one embodiment, the expressionscore is determined using the FOLR1-2.1 antibody.

In some embodiments, a score of at least 1 indicates an increasedexpression of FOLR1. In some embodiments, a score of at least 2, atleast 2 homo (>75% uniformity), or at least 2 hetero (25-75% uniformity)indicates a decreased dose of the active agent should be administered.In some embodiments, the cancer is lung cancer or endometrial cancer. Insome embodiments, a score of at least 3, at least 3 homo (>75%uniformity), or at least 3 hetero (25-75% uniformity) indicates adecreased dose of the active agent should be administered. In someembodiments, the cancer is lung cancer, endometrial cancer, or ovariancancer.

In some embodiments, a score of at least 1 indicates an increasedexpression of FOLR1. In some embodiments, a score of at least 2, atleast 2 homo (>75% uniformity), or at least 2 hetero (25-75% uniformity)identifies the cancer as likely to respond to a low dose anti-FOLR1treatment. In some embodiments, the cancer is lung cancer or endometrialcancer. In some embodiments, a score of at least 3, at least 3 homo(>75% uniformity), or at least 3 hetero (25-75% uniformity) identifiesthe cancer as likely to respond to a low dose anti-FOLR1 treatment. Insome embodiments, the cancer is lung cancer, endometrial cancer, orovarian cancer.

In some embodiments, a score of at least 2, at least 2 homo (>75%uniformity), or at least 2 hetero (25-75% uniformity) identifies thecancer as being sensitive to treatment with an active agent comprisingan anti-FOLR1 antibody or antigen-binding fragment thereof. In someembodiments, the cancer is lung cancer or endometrial cancer. In someembodiments, a score of at least 3, at least 3 homo (>75% uniformity),or at least 3 hetero (25-75% uniformity) identifies the cancer as beingsensitive to treatment with an active agent comprising an anti-FOLR1antibody or antigen-binding fragment thereof. In some embodiments, thecancer is lung cancer, endometrial cancer, or ovarian cancer.

In some embodiments, an H-score of at least 50 identifies a cancer asbeing sensitive to treatment with an active agent comprising ananti-FOLR1 antibody or antigen-binding fragment thereof. In someembodiments, an H-score of at least 75 identifies an ovarian cancer asbeing sensitive to treatment with an active agent comprising ananti-FOLR1 antibody or antigen-binding fragment thereof. In someembodiments, an H-score of at least 50 identifies an NSCLC as beingsensitive to treatment with an active agent comprising an anti-FOLR1antibody or antigen-binding fragment thereof. In some embodiments, anH-score of at least 50 identifies an endometrial cancer as beingsensitive to treatment with an active agent comprising an anti-FOLR1antibody or antigen-binding fragment thereof. In one embodiment, anH-score is determined using the FOLR1-2.1 antibody.

In some embodiments, at least 25% of FOLR1 membrane expression in aovarian tumor sample with an intensity of at least 3 identifies thecancer as being sensitive to treatment with an active agent comprisingan anti-FOLR1 antibody or antigen-binding fragment thereof. In someembodiments, at least 25% of FOLR1 membrane expression in an NSCLCsample with an intensity of at least 2 identifies the cancer as beingsensitive to treatment with an active agent comprising an anti-FOLR1antibody or antigen-binding fragment thereof. In some embodiments, atleast 25% of FOLR1 membrane expression in an endometrial tumor samplewith an intensity of at least 2 identifies the cancer as being sensitiveto treatment with an active agent comprising an anti-FOLR1 antibody orantigen-binding fragment thereof. In one embodiment, the expressionscore is determined using the FOLR1-2.1 antibody.

In some embodiments, the reference sample is a positive reference sampleor a negative reference sample. In some embodiments, the referencesample comprises cells, cell pellets, or tissue.

In some embodiments, the antibody, antigen-binding fragment thereof, orpolypeptide of comprises a detection reagent selected from the groupconsisting of: an enzyme, a fluorophore, a radioactive label, and aluminophore. In some embodiments, the detection reagent is selected fromthe group consisting of: biotin, digoxigenin, fluorescein, tritium, andrhodamine.

In some embodiments, the cancer is a FOLR1 positive cancer. In someembodiments, the cancer is selected from the group consisting ofovarian, brain, breast, uterine, endometrial, pancreatic, renal, andlung cancer. In some embodiments, the lung cancer is non small cell lungcancer or bronchioloalveolar carcinoma. In some embodiments, the ovariancancer is epithelial ovarian cancer. In some embodiments, the ovariancancer is platinum resistant, relapsed, or refractory.

In some embodiments, FOLR1 expression is detected using at least oneadditional anti-FOLR1 antibody or antigen-binding fragment thereof. Insome embodiments, FOLR1 expression is measured using two anti-FOLR1antibodies or antigen-binding fragments thereof. In some embodiments, atleast one antibody or antigen-binding fragment thereof is bound to asolid support. In some embodiments, at least one antibody orantigen-binding fragment thereof is bound to a microtiter plate.

In some embodiments, at least one additional antibody or antigen-bindingfragment thereof comprises a detection agent. In some embodiments, thedetection agent is a chromogenic detection agent, a fluorogenicdetection agent, an enzymatic detection agent, or anelectrochemiluminescent detection agent. In some embodiments, thedetection agent is horseradish peroxidase (HRP).

In some embodiments, the ELISA is a sandwich ELISA.

In some embodiments, the active agent comprises the FOLR1 antibodyhuMov19. In some embodiments, the active agent is an antibodymaytansinoid conjugate comprising the FOLR1 antibody huMov19 (comprisinga heavy chain variable region of SEQ ID NO:45 and a light chain variableregion of SEQ ID NO:47), the maytansinoid DM4, and the cleavablesulfo-SPDB linker (IMGN853).

In some embodiments, a method for identifying a cancer as likely torespond to treatment with an antibody maytansinoid conjugate comprisingthe FOLR1 antibody huMov19, the maytansinoid DM4 and a sulfo-SPDB linker(IMGN853), comprises measuring FOLR1 using an antibody comprising aheavy chain comprising the amino acids of SEQ ID NO:27 and a light chaincomprising the amino acids of SEQ ID NO:28 in an IHC assay, wherein ascore of at least 2 hetero indicates the cancer is likely to responds tothe treatment.

In some embodiments, a method for identifying a cancer as likely torespond to treatment with an antibody maytansinoid conjugate comprisingthe FOLR1 antibody huMov19, the maytansinoid DM4 and a sulfo-SPDB linker(IMGN853), comprises measuring FOLR1 using an antibody comprising aheavy chain comprising the amino acids of SEQ ID NO:27 and a light chaincomprising the amino acids of SEQ ID NO:28 in an IHC assay, wherein ascore of at least 1 indicates the cancer is likely to responds to thetreatment.

In some embodiments, an article of manufacture provided herein comprisesa therapeutic active agent comprising an anti-FOLR1 antibody orantigen-binding fragment thereof described herein, a container, and apackage insert or label indicating that the active agent can be used totreat a cancer characterized by the increased expression of FOLR1. Insome embodiments, an article of manufacture provided herein comprises atherapeutic active agent comprising an anti-FOLR1 antibody orantigen-binding fragment thereof described herein, a container, and apackage insert or label indicating that the active agent can be used totreat a cancer characterized by the expression of FOLR1 at a level of 2,or 3 measured using an antibody, antigen-binding fragment thereof,polypeptide, or composition provided herein. In some embodiments, theanti-FOLR1 antibody of the active agent is conjugated to a cytotoxin. Insome embodiments, the package insert or label indicates that the activeagent can be used to treat a cancer characterized by the expression ofFOLR1 at a level of at least 1. In some embodiments, the package insertor label indicates that the active agent can be used to treat a cancercharacterized by the expression of FOLR1 at a level of at least 2, atleast 2 homo (>75% uniformity), or at least 2 hetero (25-75%uniformity). In some embodiments, the cancer is lung cancer orendometrial cancer. In some embodiments, the package insert or labelindicates that the active agent can be used to treat a cancercharacterized by the expression of FOLR1 at a level of at least 3, atleast 3 homo (>75% uniformity), or at least 3 hetero (25-75%uniformity). In some embodiments, the cancer is lung cancer, endometrialcancer, or ovarian cancer.

In some embodiments, a combination diagnostic and pharmaceutical kitprovided herein comprises an antibody, antigen-binding fragment thereof,polypeptide, or composition provided herein for use in diagnosis and anactive agent comprising an anti-FOLR1 antibody or antigen-bindingfragment thereof for use in therapy. In some embodiments, the detectionantibody is able to detect FOLR1 expression by IHC. In some embodiments,the detection antibody is able to detect FOLR1 expression by ELISA. Insome embodiments, the anti-FOLR1 antibody in the active agent isconjugated to a cytotoxin.

In some embodiments, a diagnostic kit provided herein comprises anantibody, antigen-binding fragment thereof or polypeptide providedherein, a reagent for immunohistochemistry (IHC), and one or morestandardized reference samples, wherein the standardized referencesamples comprise cells, cell pellets, or formalin fixed paraffinembedded tissue samples, and wherein the one or more standardizedreferenced samples are from non-FOLR1 expressing, low-FOLR1 expressing,or high FOLR1 expressing cells, cell pellets, or tissues.

In some embodiments, an immunoassay kit for detecting shed FOLR1 in asample comprises: (a) an antibody, antigen-binding fragment thereof,polypeptide, or composition provided herein, and (b) a detectionreagent. In some embodiments, the kit further comprises a solid supportfor the capture reagent. In some embodiments, the capture reagent isimmobilized on the solid support. In some embodiments, the capturereagent is coated on a microtiter plate. In some embodiments, thedetection reagent is a second FOLR1 antibody. In some embodiments, thedetection reagent is detected using a species specific antibody. In someembodiments, the kit further comprises a detection means for thedetection reagent. In some embodiments, the detection means iscolorimetric. In some embodiments, the kit further comprises a FOLR1polypeptide as an antigen standard. In some embodiments, the FOLR1polypeptide is FOLR1-Fc.

Active agents are also provided herein. In some embodiments, an activeagent comprises an anti-FOLR1 antibody or antigen-binding fragmentthereof for use in a method for treating cancer, wherein said activeagent is administered to a subject having cancer, wherein increasedexpression of FOLR1 has been detected in a cancerous sample obtainedfrom said subject using an antibody, antigen-binding fragment thereof,polypeptide or composition provided herein.

In some embodiments, an active agent comprises an anti-FOLR1 antibody orantigen-binding fragment thereof for use in a method for treatingcancer, comprising: (a) determining a FOLR1 expression score from adetection of FOLR1 expression in a cancerous sample obtained from thepatient, wherein the detection is performed using an antibody,antigen-binding fragment thereof, polypeptide, or composition providedherein; and (b) administering an active agent comprising an anti-FOLR1antibody or antigen-binding fragment thereof to the patient if the scoreindicates the patient will benefit from administration of the activeagent.

In some embodiments, an active agent comprises an anti-FOLR1 antibody orantigen-binding fragment thereof for use in a method for treatingcancer, comprising: (a) determining a FOLR1 expression score from adetection of FOLR1 expression in a cancerous sample obtained from thepatient, wherein the detection is performed using an antibody,antigen-binding fragment thereof, polypeptide, or composition providedherein; and (b) instructing a healthcare provider to administer anactive agent comprising an anti-FOLR1 antibody or antigen-bindingfragment thereof to the patient if the score indicates the patient willbenefit from administration of the active agent.

In some embodiments, an active agent comprises an anti-FOLR1 antibody orantigen-binding fragment thereof for use in a method for treatingcancer, comprising: (a) submitting a cancerous sample obtained from apatient having cancer for determining a FOLR1 expression score from adetection of FOLR1 expression using the antibody, antigen-bindingfragment thereof, polypeptide, or composition provided herein; and (b)administering an active agent comprising an anti-FOLR1 antibody orantigen-binding fragment thereof to the patient if the score indicatesthe patient will benefit from administration of the active agent.

In some embodiments, an active agent comprises an anti-FOLR1 antibody orantigen-binding fragment thereof for use in a method for treatingcancer, comprising: (a) detecting FOLR1 expression in a cancerous sampleobtained from said patient, wherein the detection is performed using theantibody, antigen-binding fragment thereof, polypeptide, or compositionprovided herein; (b) determining a FOLR1 expression score for saidcancerous sample; and (c) administering an active agent comprising ananti-FOLR1 antibody or antigen-bidning fragment thereof to the patientif the score indicates the patient will benefit from administration ofthe active agent.

In some embodiments, an active agent comprises an anti-FOLR1 antibody orantigen-binding fragment thereof for use in a method for treatingcancer, comprising: (a) administering to a patient a fixed dose of anactive agent comprising an anti-FOLR1 antibody or antigen-bindingfragment thereof; (b) detecting the FOLR1 expression level in acancerous sample obtained from the patient relative to the FOLR1 levelin a reference sample, wherein the detection is performed using anantibody, antigen-binding fragment thereof, polypeptide, or compositionprovided herein; and (c) increasing the amount or frequency ofsubsequent fixed doses if the patient's FOLR1 level is elevated.

In some embodiments, an active agent comprises an anti-FOLR1 antibody orantigen-binding fragment thereof for use in a method for treatingcancer, comprising the step of optimizing the therapeutic regimen ofsaid active agent comprising: (a) administering an increased dose of anactive agent comprising an anti-FOLR1 antibody or antigen-bindingfragment thereof to a subject having cancer wherein an increasedexpression of FOLR1 in a cancerours sample from said subject has beendetected using the antibody, antigen-binding fragment thereof,polypeptide, or composition provided herein; or (b) administering adecreased dose of the active agent to a subject having cancer wherein adecreased expression of FOLR1 in a cancerous sample from said subjecthas been detected.

In some embodiments, an active agent comprises an anti-FOLR1 antibody orantigen-binding fragment thereof for use in a method for treatingcancer, comprising the step of optimizing the therapeutic regimen ofsaid active agent comprising: (a) detecting the level of FOLR1expression in a cancerous sample from said subject using an antibody,antigen-binding fragment thereof, polypeptide, or composition providedherein; (b) determining a FOLR1 expression score for said canceroussample; and (c) administering an increased dose of an active agentcomprising an anti-FOLR1 antibody or antigen-binding fragment thereof tothe subject if the score is low or administering a decreased dose of theactive agent to the subject if the score is high.

In some embodiments, an active agent comprises an anti-FOLR1 antibody orantigen-binding fragment thereof for use in a method for treatingcancer, wherein FOLR1-expressing cancer cells in a cancer patient aredecreased, wherein: (a) the FOLR1 level in a cancerous sample obtainedfrom a patient is detected by comparing it to the FOLR1 level in areference sample using an antibody, antigen-binding fragment thereof,polypeptide, or composition provided herein; and (b) a fixed dose of theactive agent is administered to the patient if the patient's FOLR1 levelis elevated; wherein the administration of the active agent decreasesthe number of FOLR1-expressing cancer cells in the patient.

In some embodiments, an active agent comprises an anti-FOLR1 antibody orantigen-binding fragment thereof for use in a method for treatingcancer, wherein FOLR1-expressing cancer cells in a cancer patient aredecreased, wherein: (a) a fixed dose of the active agent is administeredto a patient having a cancer; (b) the FOLR1 level in a cancerous sampleobtained from the patient is detected relative to the FOLR1 level in areference sample using an antibody, antigen-binding fragment thereof,polypeptide, or composition provided herein; and (c) the amount orfrequency of subsequent fixed doses is increased if the patient's FOLR1level is elevated compared to the reference sample; wherein theadministration of the active agent decreases the number ofFOLR1-expressing cancer cells in the patient.

Anti-FOLR1 antibodies and antigen-binding fragments thereof for uses ismethods of monitoring and methods of diagnosing are also providedherein. In some embodiments, an anti-FOLR1 antibody or antigen-bindingfragment thereof for use in a method for monitoring the therapeuticefficacy of a fixed dose of the active agent in a patient comprises: (a)detecting a first FOLR1 level in a biological sample from a patienthaving cancer using an antibody, antigen-binding fragment thereof,polypeptide, or composition provided herein; (b) administering to thepatient a fixed dose of the active agent; (c) detecting a second FOLR1level in a biological sample from the patient following active agentadministration, wherein the detecting is performed using an antibody,antigen-binding fragment thereof, polypeptide, or composition providedherein; and (d) comparing the second FOLR1 level to the first FOLR1level; wherein a decrease between the first and second FOLR1 levelindicates therapeutic efficacy.

In some embodiments, an anti-FOLR1 antibody or antigen-binding fragmentthereof for use in a method for diagnosing whether a subject havingcancer is likely to respond to a low dose anti-FOLR1 treatment regimen,comprises: (a) contacting a biological sample comprising cells from saidcancer with an antibody, antigen-binding fragment thereof, polypeptide,or composition provided herein; (b) detecting binding of said antibody,antigen-binding fragment, or polypeptide to said biological sample of(a); (c) assigning a score to said binding of step (b), wherein saidscore is assigned based on comparison to one or more reference samples;and (d) comparing said score in step (c) to the score of a referencetissue or cell, wherein a score for said cancer FOLR1 level that isgreater than the score for a normal or low FOLR1 expressing referencesample or a score for said cancer FOLR1 level that is equal to orgreater than the score for a high FOLR1 expressing reference sampleidentifies said cancer as likely to respond to a low dose of an activeagent comprising an anti-FOLR1 antibody or antigen-binding fragmentthereof.

In some embodiments, an anti-FOLR1 antibody or antigen-binding fragmentthereof for use in a method for diagnosing whether a cancer is sensitiveto treatment with an anti-FOLR1 treatment, comprises: (a) detecting thelevel of FOLR1 expression in a cancerous sample from said cancer usingan antibody, antigen-binding fragment thereof, polypeptide, orcomposition provided herein, wherein said detecting comprises the use ofa method that distinguishes between staining intensity or staininguniformity in a FOLR1 expressing cancerous sample as compared tostaining intensity or staining uniformity in one or more referencesamples; (b) determining a FOLR1 staining intensity or staininguniformity score for said cancerous sample; and (c) comparing the FOLR1staining intensity or staining uniformity score determined in step (b)to a relative value determined by measuring FOLR1 protein expression inat least one reference sample, wherein said at least one referencesample is a tissue, cell, or cell pellet sample which is not sensitiveto treatment with an active agent comprising an anti-FOLR1 antibody orantigen-binding fragment thereof and wherein a FOLR1 staining intensityscore for said cancerous sample determined in step (b) that is higherthan said relative value identifies said cancer as being sensitive totreatment with the active agent.

In some embodiments, an anti-FOLR1 antibody or antigen-binding fragmentthereof for use in a method for diagnosing whether a cancer is sensitiveto treatment with an anti-FOLR1 treatment, comprising: (a) detecting thelevel of FOLR1 expression in a cancerous sample from said cancer usingan antibody, antigen-binding fragment thereof, polypeptide, orcomposition provided herein, wherein said detecting comprises the use ofa method that distinguishes between staining intensity or staininguniformity in a FOLR1 expressing cancerous sample as compared tostaining intensity or staining uniformity in one or more referencesamples; (b) determining a FOLR1 staining intensity or staininguniformity score for said cancerous sample; and (c) comparing the FOLR1staining intensity or staining uniformity score determined in step (b)to a relative value determined by measuring FOLR1 protein expression inat least one reference sample, wherein said at least one referencesample is a tissue, cell, or cell pellet sample which is sensitive totreatment with an active agent comprising an anti-FOLR1 antibody orantigen-binding fragment thereof and wherein a FOLR1 staining intensityscore for said cancerous sample determined in step (b) that is higherthan said relative value identifies said cancer as being sensitive totreatment with the active agent.

In some embodiments, the use of the active agents or anti-FOLR1antibodies or antigen-binding fragments thereof further comprisesadministering an active agent comprising an anti-FOLR1 antibody orantigen-fragment thereof to the subject from whom the cancerous sampleor biological sample was obtained.

In some embodiments, the cancerous sample or biological sample is abodily fluid, cell, or tissue sample. In some embodiments, the cell is acirculating tumor cell. In some embodiments, the bodily fluid is blood,ascites, urine, plasma, serum, or peripheral blood.

In some embodiments of the active agents or anti-FOLR1 antibodies orantigen-binding fragments thereof, the detecting is by enzyme linkedimmunosorbent assay (ELISA) and/or by immunohistochemistry (IHC). Insome embodiments, the IHC is calibrated IHC that can distinguishdifferent levels of FOLR1 expression. In some embodiments, the IHCproduces a range of staining intensity for samples having low cellsurface FOLR1 expression, intermediate FOLR1 cell surface expression, orhigh FOLR1 cell surface expression. In some embodiments, the IHCdistinguishes between staining intensity and staining uniformity in aFOLR1 expressing cancerous sample or biological sample as compared to areference sample. In some embodiments, IHC is performed manually. Insome embodiments, the IHC is performed using an automated system. Insome embodiments, a FOLR1 score is determined from the IHC. In someembodiments, the IHC with an antibody or antigen-binding fragmentdescribed herein produces a range of staining for cells that haveincreased FOLR1 expression, particularly those within the level ofstaining equal to or greater than 2.

In some embodiments, a score of at least 2 indicates that the patientwill benefit from administration of an active agent comprising ananti-FOLR1 antibody or antigen-binding fragment thereof. In someembodiments, a score of at least 2 homo (>75% uniformity) or at least 2hetero (25-75% uniformity) indicates that the patient will benefit fromadministration of an active agent comprising an anti-FOLR1 antibody orantigen-binding fragment thereof. In some embodiments, the cancer islung cancer or endometrial cancer.

In some embodiments, a score of at least 3 indicates that the patientwill benefit from administration of an active agent comprising ananti-FOLR1 antibody or antigen-binding fragment thereof In someembodiments, a score of at least 3 homo (>75% uniformity) or at least 3hetero (25-75% uniformity) indicates that the patient will benefit fromadministration of an active agent comprising an anti-FOLR1 antibody orantigen-binding fragment thereof. In some embodiments, the cancer islung cancer, endometrial cancer, or ovarian cancer.

In some embodiments, a score of at least 2 indicates that the patientwill benefit from administration of an active agent comprising ananti-FOLR1 antibody or antigen-binding fragment thereof In someembodiments, a score of at least 2 homo (>75% uniformity) or at least 2hetero (25-75% uniformity) indicates that the patient will benefit fromadministration of an active agent comprising an anti-FOLR1 antibody orantigen-binding fragment thereof. In some embodiments, the cancer islung cancer, endometrial cancer, or ovarian cancer.

In some embodiments, a score of at least 2 indicates an a decreased doseof the active agent should be administered. In some embodiments, a scoreof at least 2 homo (>75% uniformity) or at least 2 hetero (25-75%uniformity) indicates an a decreased dose of the active agent should beadministered. In some embodiments, the cancer is lung cancer,endometrial cance, or ovarian cancer.

In some embodiments, a score of at least 2 identifies the cancer aslikely to respond to a low dose anti-FOLR1 treatment. In someembodiments, a score of at least 2 homo (>75% uniformity) or 2 hetero(25-75% uniformity) identifies the cancer as likely to respond to a lowdose anti-FOLR1 treatment. In some embodiments, the cancer is lungcancer or endometrial cancer.

In some embodiments, a score of at least 3 identifies the cancer aslikely to respond to a low dose anti-FOLR1 treatment. In someembodiments, a score of at least 3 homo (>75% uniformity) or at least 3hetero (25-75% uniformity) identifies the cancer as likely to respond toa low dose anti-FOLR1 treatment. In some embodiments, the cancer is lungcancer, endometrial cance, or ovarian cancer.

In some embodiments, a score of at least 2 identifies the cancer aslikely to respond to a low dose anti-FOLR1 treatment. In someembodiments, a score of at least 2 homo (>75% uniformity) or at least 2hetero (25-75% uniformity) identifies the cancer as likely to respond toa low dose anti-FOLR1 treatment. In some embodiments, the cancer is lungcancer, endometrial cance, or ovarian cancer.

In some embodiments, a score of at least 2 identifies the cancer asbeing sensitive to treatment with an active agent comprising ananti-FOLR1 antibody or antigen-binding fragment thereof. In someembodiments, a score of at least 2 homo (>75% uniformity) or at least 2hetero (25-75% uniformity) identifies the cancer as being sensitive totreatment with an active agent comprising an anti-FOLR1 antibody orantigen-binding fragment thereof. In some embodiments, the cancer islung cancer or endometrial cancer.

In some embodiments, a score of at least 3 identifies the cancer asbeing sensitive to treatment with an active agent comprising ananti-FOLR1 antibody or antigen-binding fragment thereof. In someembodiments, a score of at least 3 homo (>75% uniformity) or at least 3hetero (25-75% uniformity) identifies the cancer as being sensitive totreatment with an active agent comprising an anti-FOLR1 antibody orantigen-binding fragment thereof. In some embodiments, the cancer islung cancer, endometrial cance, or ovarian cancer.

In some embodiments, a score of at least 2 identifies the cancer asbeing sensitive to treatment with an active agent comprising ananti-FOLR1 antibody or antigen-binding fragment thereof. In someembodiments, a score of at least 2 homo (>75% uniformity) or at least 2hetero (25-75% uniformity) identifies the cancer as being sensitive totreatment with an active agent comprising an anti-FOLR1 antibody orantigen-binding fragment thereof. In some embodiments, the cancer islung cancer, endometrial cance, or ovarian cancer.

In some embodiments, a reference sample is a positive reference sampleor a negative reference sample. In some embodiments, the referencesample comprises cells, cell pellets, or tissue.

In some embodiments of the active agent or anti-FOLR1 antibody orantigen-binding fragment thereof for a use provided herein, theantibody, antigen-binding fragment, or polypeptide provided hereinfurther comprises a detection reagent selected from the group consistingof: an enzyme, a fluorophore, a radioactive label, and a luminophore. Insome embodiments, the detection reagent is selected from the groupconsisting of: biotin, digoxigenin, fluorescein, tritium, and rhodamine.

In some embodiments of the active agent or anti-FOLR1 antibody orantigen-binding fragment thereof for a use provided herein, the canceris a FOLR1 positive cancer. In some embodiments, the cancer is selectedfrom the group consisting of ovarian, brain, breast, uterine,endometrial, pancreatic, renal, and lung cancer. In some embodiments,the lung cancer is non small cell lung cancer or bronchioloalveolarcarcinoma. In some embodiments, the ovarian cancer is epithelial ovariancancer. In some embodiments, the ovarian cancer is platinum resistant,relapsed, or refractory.

In some embodiments of the active agent or anti-FOLR1 antibody orantigen-binding fragment thereof for a use provided herein, the FOLR1expression is detected using at least one additional anti-FOLR1 antibodyor antigen-binding fragment thereof. In some embodiments, the FOLR1expression is measured using two anti-FOLR1 antibodies orantigen-binding fragments thereof. In some embodiments, at least oneantibody or antigen-binding fragment thereof is bound to a solidsupport. In some embodiments, at least one antibody or antigen-bindingfragment thereof is bound to a microtiter plate. In some embodiments, atleast one antibody or antigen-binding fragment thereof comprises adetection agent. In some embodiments, the detection agent is achromogenic detection agent, a fluorogenic detection agent, an enzymaticdetection agent, or an electrochemiluminescent detection agent. In someembodiments, the detection agent is horseradish peroxidase (HRP). Insome embodiments, the ELISA is a sandwich ELISA.

In some embodiments of the active agent or anti-FOLR1 antibody orantigen-binding fragment thereof for a use provided herein, the activeagent comprises the FOLR1 antibody huMov19 or is the FOLR1 antibodyhuMov19. In some embodiments, the active agent is administered as anantibody maytansinoid conjugate further comprising the maytansinoid DM4and the cleavable sulfo-SPDB linker (IMGN853).

In some embodiments, an antibody, antigen-binding fragment, polypeptide,or composition provided herein is for use as a diagnostic.

In some embodiments, an antibody, antigen-binding fragment, polypeptide,or composition provided herein is for use in a method for diagnosingcancer in a patient suffering therefrom. In some embodiments, the canceris associated with elevated levels of FOLR1.

In some embodiments, the binding affinity of an antibody,antigen-binding fragment, or polypeptide is a binding affinity obtainedin Example 3 and/or shown in FIGS. 4, 5, and/or 6.

BRIEF DESCRIPTIONS OF THE DRAWINGS

FIG. 1 provides images of IHC staining of NSCLC and ovarian endometrioidadenocarcinoma samples using the 353.2-1 and 353.9-20 antibodies.

FIG. 2 provides images of IHC staining of normal salivary gland andpancreas samples using the 353.2-1 and 353.9-20 antibodies.

FIG. 3 provides images of Western blots of cell lysates using the353.9-21, 353.2-1, 353.3-8, and 353.5-7 antibodies.

FIGS. 4A and 4B show the binding of 353.2-1, 353.3-1, 353.5-7, and353.9-21 antibodies to denatured KB cells (A) and non-denatured T47Dcells (B) using a fluorescence activated cell sorter (FACS).

FIG. 5 shows the binding of 353.2-1, 353.3-1, 353.5-7, and 353.9-21antibodies to recombinant human FOLR1 using ELISA.

FIGS. 6A and 6B show the binding of an anti-FOLR2 antibody and 353.2-1,353.3-1, 353.5-7, and 353.9-21 antibodies to FOLR2 (A) and the bindingof an anti-FOLR3 antibody and 353.2-1, 353.3-1, 353.5-7, and 353.9-21antibodies to FOLR3 (B) by ELISA.

FIG. 7 shows the binding of anti-FOLR1 antibodies 2.1 and huMov19 todeglycosylated and non-treated recombinant human FOLR1 by ELISA.

FIG. 8 shows the binding of anti-FOLR1 antibodies 2.1, huMov19, andBN3.2 to deglysolyated and non-treated lysates of KB and Igrov-1 cellsby western blot analysis.

FIGS. 9A and 9B show the relevant amino acids for resurfacing of theanti-FOLR1 FRIHC2-1 antibody and the kabat position corresponding toeach residue.

FIGS. 10A and 10B show the alignment of murine and humanized FRIHC2-1antibody sequences for resurfacing. The murine heavy and light chainsequences correspond to SEQ ID NO:27 and SEQ ID NO:28, respectively. Theresurfaced humanized heavy chain sequence corresponds to SEQ ID NO: 62,and the resurfaced human light chain version 1.0 and version 1.1sequences correspond to SEQ ID NO:63 and SEQ ID NO:64, respectively. Theleader “S” in the light chain sequence (framework position −1) is notconsidered for humanization and is not used in the humanized antibodysequence, so it is not shown in the figure.

FIGS. 11A and 11B show the relevant amino acids for CDR grafting of theanti-FOLR1 FRIHC2-1 antibody and the kabat position corresponding toeach residue.

FIGS. 12A and 12B show the alignment of murine and humanized FRIHC2-1sequences for CDR grafting. The murine heavy and light chain sequencescorrespond to SEQ ID NO:27 and SEQ ID NO:28, respectively. The graftedhumanized heavy chain sequence corresponds to SEQ ID NO: 65, and thegrafted human light chain version 1.0 and version 1.1 sequencescorrespond to SEQ ID NO:66 and SEQ ID NO:67, respectively. The leader“S” in the light chain sequence (framework position −1) is notconsidered for humanization and is not used in the humanized antibodysequence, so it is not shown in the figure.

FIG. 13 provides images of IHC staining of lung adenocarcinoma tissuesusing the FOLR1-2.1 (353-2.1) antibody at varying dilutions.

FIGS. 14A, 14B, and 14C provide images of IHC staining of positivenormal tissue (fallopian tube) (A) and cells (FOLR1 transfected cells(B)) and negative cells (untransfected cells (C)) using the FOLR1-2.1(353-2.1) antibody.

FIGS. 15A and 15B provide images of IHC staining of ovarian cancertissue (A) and lung adenocarcinoma tissue (B) samples using theFOLR1-2.1 (353-2.1) antibody.

FIG. 16 shows membrane staining of tumor cells in an endometrial cancersample with the FOLR1-2.1 assay. The stromal cells are not stained.

FIG. 17 shows a comparison of staining and scoring difference between(A) the FOLR1-2.1 assay and (B) the BN3.2 assay.

DETAILED DESCRIPTION OF THE INVENTION

The present disclosure provides methods of detecting human folatereceptor 1 (FOLR1), including membrane FOLR1, shed FOLR1, and FOLR1 oncirculating tumor cells, and improving the efficacy of or the likelihoodof response to the treatment of cancers characterized by theoverexpression of FOLR1. The detection methods can detect a clinicallyrelevant dynamic range of FOLR1 and therefore can be used for patientstratification, to monitor or determine therapeutic efficacy, or thelikelihood of response to the treatment of cancers characterized by theover expression of FOLR1. Novel FOLR1-binding polypeptides, such asantibodies, that are useful in the FOLR1 detection methods (e.g., IHCfor membrane bound and cell associated FOLR1) are also disclosed.Related polypeptides and polynucleotides, compositions comprising theFOLR1-binding agents, and methods of making the FOLR1-binding agents arealso provided.

I. Definitions

To facilitate an understanding of the present invention, a number ofterms and phrases are defined below.

The terms “human folate receptor 1,” “FOLR1,” or “folate receptor alpha(FR-α)”, as used herein, refers to any native human FOLR1, unlessotherwise indicated. Thus, all of these terms can refer to either aprotein or nucleic acid sequence as indicated herein. The term “FOLR1”encompasses “full-length,” unprocessed FOLR1 as well as any form ofFOLR1 that results from processing within the cell. The term alsoencompasses naturally occurring variants of FOLR1 protein or nucleicacid, e.g., splice variants, allelic variants and isoforms. The FOLR1polypeptides and polynucleotides described herein can be isolated from avariety of sources, such as from human tissue types or from anothersource, or prepared by recombinant or synthetic methods. Examples ofFOLR1 sequences include, but are not limited to NCBI reference numbersP15328, NP_001092242.1, AAX29268.1, AAX37119.1. NP_057937.1, andNP_057936.1.

The terms “shed antigen” and “shed FOLR1” are used interchangeablyherein. These terms refer to a FOLR1 protein that is soluble and that isnot cell associated. In some embodiments it includes the extracellulardomain (ECD) and the glycosylphosphatidyl inositol (GPI) linker. In oneembodiment, the shed FOLR1 includes only the ECD. FOLR1 protein includesa signal peptide (amino acids 1-24), the FOLR1 protein chain (aminoacids 25-233 or 234), and a propeptide which can be cleaved (amino acids235 to 257). Mature FOLR1 protein lacks the signal peptide. Shed FOLR1can include amino acids 1 to 257, 1 to 233, 1 to 234, 25 to 233, 25 to234, or any other fragments thereof. In some embodiments the signalsequence is cleaved. In other embodiments the ECD and the GPI portioncan be embedded in a membrane (e.g., a soluble lipid raft). In oneembodiment, the shed FOLR1 can include amino acids 1-233 or a fragmentthereof.

The term “antibody” means an immunoglobulin molecule that recognizes andspecifically binds to a target, such as a protein, polypeptide, peptide,carbohydrate, polynucleotide, lipid, or combinations of the foregoingthrough at least one antigen recognition site within the variable regionof the immunoglobulin molecule. As used herein, the term “antibody”encompasses intact polyclonal antibodies, intact monoclonal antibodies,antibody fragments (such as Fab, Fab′, F(ab′)2, and Fv fragments),single chain Fv (scFv) mutants, multispecific antibodies such asbispecific antibodies, chimeric antibodies, humanized antibodies, humanantibodies, fusion proteins comprising an antigen determination portionof an antibody, and any other modified immunoglobulin moleculecomprising an antigen recognition site so long as the antibodies exhibitthe desired biological activity. An antibody can be of any of the fivemajor classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, orsubclasses (isotypes) thereof (e.g., IgG1, IgG2. IgG3, IgG4, IgA1 andIgA2), based on the identity of their heavy-chain constant domainsreferred to as alpha, delta, epsilon, gamma, and mu, respectively. Thedifferent classes of immunoglobulins have different and well knownsubunit structures and three-dimensional configurations. Antibodies canbe naked or conjugated to other molecules such as toxins, radioisotopes,etc.

In some embodiments, an antibody is a non-naturally occurring antibody.In some embodiments, an antibody is purified from natural components. Insome embodiments, an antibody is recombinantly produced. In someembodiments, an antibody is produced by a hybridoma.

A “blocking” antibody or an “antagonist” antibody is one which inhibitsor reduces biological activity of the antigen it binds, such as FOLR1.In a certain embodiment, blocking antibodies or antagonist antibodiessubstantially or completely inhibit the biological activity of theantigen. Desirably, the biological activity is reduced by 10%, 20%, 30%,50%, 70%, 80%, 90%, 95%, or even 100%.

The term “anti-FOLR1 antibody” or “an antibody that binds to FOLR1”refers to an antibody that is capable of binding FOLR1 with sufficientaffinity such that the antibody is useful as a diagnostic and/ortherapeutic agent in targeting FOLR1. Unless otherwise specified, theextent of binding of an anti-FOLR1 antibody to an unrelated, non-FOLR1protein is less than about 10% of the binding of the antibody to FOLR1as measured, e.g., by a radioimmunoassay (RIA). In certain embodiments,an antibody that binds to FOLR1 has a dissociation constant (Kd) of ≤1μM, ≤100 nM, ≤10 nM, ≤1 nM, or ≤0.1 nM. In one embodiment, theanti-FOLR1 antibody does not bind FOLR2, FOLR3, FOLR4, or folic acid.Examples of FOLR1 antibodies are known in the art and are disclosed inU.S. Published Application Nos. 2012/0009181 and 2012/0282175 and U.S.Provisional Application Nos. 61/695,791 and 61/756,254, and PCTpublication WO2011/106528, each of which is herein incorporated byreference. The sequences of anti-FOLR1 antibodies and antigen-bindingfragments thereof are provided in Tables 1-8.

The term “antibody fragment” refers to a portion of an intact antibodyand refers to the antigenic determining variable regions of an intactantibody. Examples of antibody fragments include, but are not limitedto, Fab, Fab′, F(ab′)2, and Fv fragments, linear antibodies, singlechain antibodies, and multispecific antibodies formed from antibodyfragments. The term “antigen-binding fragment” of an antibody includesone or more fragments of an antibody that retain the ability tospecifically bind to an antigen. It has been shown that theantigen-binding function of an antibody can be performed by certainfragments of a full-length antibody. Examples of binding fragmentsencompassed within the term “antigen-binding fragment” of an antibodyinclude (without limitation): (i) a Fab fragment, a monovalent fragmentconsisting of the VL, VH, CL and CH1 domains (e.g., an antibody digestedby papain yields three fragments: two antigen-binding Fab fragments, andone Fc fragment that does not bind antigen); (ii) a F(ab′)₂ fragment, abivalent fragment comprising two Fab fragments linked by a disulfidebridge at the hinge region (e.g., an antibody digested by pepsin yieldstwo fragments: a bivalent antigen-binding F(ab′)₂ fragment, and a pFc′fragment that does not bind antigen) and its related F(ab′) monovalentunit; (iii) a Fd fragment consisting of the VH and CH1 domains (i.e.,that portion of the heavy chain which is included in the Fab); (iv) a Fvfragment consisting of the VL and VH domains of a single arm of anantibody, and the related disulfide linked Fv; (v) a dAb (domainantibody) or sdAb (single domain antibody) fragment (Ward et al., Nature341:544-546, 1989), which consists of a VH domain; and (vi) an isolatedcomplementarity determining region (CDR).

A “monoclonal antibody” refers to a homogeneous antibody populationinvolved in the highly specific recognition and binding of a singleantigenic determinant, or epitope. This is in contrast to polyclonalantibodies that typically include different antibodies directed againstdifferent antigenic determinants. The term “monoclonal antibody”encompasses both intact and full-length monoclonal antibodies as well asantibody fragments (such as Fab, Fab′, F(ab′)2, Fv), single chain (scFv)mutants, fusion proteins comprising an antibody portion, and any othermodified immunoglobulin molecule comprising an antigen recognition site.Furthermore, “monoclonal antibody” refers to such antibodies made in anynumber of manners including but not limited to by hybridoma, phageselection, recombinant expression, and transgenic animals.

The term “humanized antibody” refers to forms of non-human (e.g.,murine) antibodies that are specific immunoglobulin chains, chimericimmunoglobulins, or fragments thereof that contain minimal non-human(e.g., murine) sequences. Typically, humanized antibodies are humanimmunoglobulins in which residues from the complementary determiningregion (CDR) are replaced by residues from the CDR of a non-humanspecies (e.g., mouse, rat, rabbit, hamster) that have the desiredspecificity, affinity, and capability (Jones et al., 1986, Nature,321:522-525; Riechmann et al., 1988, Nature, 332:323-327; Verhoeyen etal., 1988, Science, 239:1534-1536). In some instances, the Fv frameworkregion (FR) residues of a human immunoglobulin are replaced with thecorresponding residues in an antibody from a non-human species that hasthe desired specificity, affinity, and capability. The humanizedantibody can be further modified by the substitution of additionalresidues either in the Fv framework region and/or within the replacednon-human residues to refine and optimize antibody specificity,affinity, and/or capability. In general, the humanized antibody willcomprise substantially all of at least one, and typically two or three,variable domains containing all or substantially all of the CDR regionsthat correspond to the non-human immunoglobulin whereas all orsubstantially all of the FR regions are those of a human immunoglobulinconsensus sequence. The humanized antibody can also comprise at least aportion of an immunoglobulin constant region or domain (Fc), typicallythat of a human immunoglobulin. Examples of methods used to generatehumanized antibodies are described in U.S. Pat. Nos. 5,225,539 and5,639,641, Roguska et al., Proc. Natl. Acad. Sci., USA, 91(3):969-973(1994), and Roguska et al., Protein Eng. 9(10):895-904 (1996). In someembodiments, a “humanized antibody” is a resurfaced antibody. In someembodiments, a “humanized antibody” is a CDR-grafted antibody.

A “variable region” of an antibody refers to the variable region of theantibody light chain or the variable region of the antibody heavy chain,either alone or in combination. The variable regions of the heavy andlight chain each consist of four framework regions (FR) connected bythree complementarity determining regions (CDRs) also known ashypervariable regions. The CDRs in each chain are held together in closeproximity by the FRs and, with the CDRs from the other chain, contributeto the formation of the antigen-binding site of antibodies. There are atleast two techniques for determining CDRs; (1) an approach based oncross-species sequence variability (i.e., Kabat et al. Sequences ofProteins of Immunological Interest, (5th ed., 1991, National Institutesof Health, Bethesda Md.)); and (2) an approach based on crystallographicstudies of antigen-antibody complexes (Al-lazikani et al (1997) J.Molec. Biol. 273:927-948)). In addition, combinations of these twoapproaches are sometimes used in the art to determine CDRs.

The Kabat numbering system is generally used when referring to a residuein the variable domain (approximately residues 1-107 of the light chainand residues 1-113 of the heavy chain) (e.g., Kabat et al., Sequences ofImmunological Interest, 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda. Md. (1991)).

The amino acid position numbering as in Kabat, refers to the numberingsystem used for heavy chain variable domains or light chain variabledomains of the compilation of antibodies in Kabat et al., Sequences ofProteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991). Using thisnumbering system, the actual linear amino acid sequence can containfewer or additional amino acids corresponding to a shortening of, orinsertion into, a FR or CDR of the variable domain. For example, a heavychain variable domain can include a single amino acid insert (residue52a according to Kabat) after residue 52 of H2 and inserted residues(e.g., residues 82a, 82b, and 82c, etc. according to Kabat) after heavychain FR residue 82. The Kabat numbering of residues can be determinedfor a given antibody by alignment at regions of homology of the sequenceof the antibody with a “standard” Kabat numbered sequence. Chothiarefers instead to the location of the structural loops (Chothia and LeskJ. Mol. Biol. 196:901-917 (1987)). The end of the Chothia CDR-H1 loopwhen numbered using the Kabat numbering convention varies between H32and H34 depending on the length of the loop (this is because the Kabatnumbering scheme places the insertions at H35A and H35B; if neither 35Anor 35B is present, the loop ends at 32; if only 35A is present, theloop ends at 33; if both 35A and 35B are present, the loop ends at 34).The AbM hypervariable regions represent a compromise between the KabatCDRs and Chothia structural loops, and are used by Oxford Molecular'sAbM antibody modeling software.

Loop Kabat AbM Chothia L1 L24-L34 L24-L34 L24-L34 L2 L50-L56 L50-L56L50-L56 L3 L89-L97 L89-L97 L89-L97 H1 H31-H35B H26-H35B H26-H32 . . . 34(Kabat Numbering) H1 H31-H35 H26-H35 H26-H32 (Chothia Numbering) H2H50-H65 H50-H58 H52-H56 H3 H95-H102 H95-H102 H95-H102

The term “human antibody” means an antibody produced by a human or anantibody having an amino acid sequence corresponding to an antibodyproduced by a human made using any technique known in the art. Thisdefinition of a human antibody includes intact or full-lengthantibodies, fragments thereof, and/or antibodies comprising at least onehuman heavy and/or light chain polypeptide such as, for example, anantibody comprising murine light chain and human heavy chainpolypeptides.

The term “chimeric antibodies” refers to antibodies wherein the aminoacid sequence of the immunoglobulin molecule is derived from two or morespecies. Typically, the variable region of both light and heavy chainscorresponds to the variable region of antibodies derived from onespecies of mammals (e.g., mouse, rat, rabbit, etc.) with the desiredspecificity, affinity, and capability while the constant regions arehomologous to the sequences in antibodies derived from another (usuallyhuman) to avoid eliciting an immune response in that species.

The terms “epitope” or “antigenic determinant” are used interchangeablyherein and refer to that portion of an antigen capable of beingrecognized and specifically bound by a particular antibody. When theantigen is a polypeptide, epitopes can be formed both from contiguousamino acids and noncontiguous amino acids juxtaposed by tertiary foldingof a protein. Epitopes formed from contiguous amino acids are typicallyretained upon protein denaturing, whereas epitopes formed by tertiaryfolding are typically lost upon protein denaturing. An epitope typicallyincludes at least 3, and more usually, at least 5 or 8-10 amino acids ina unique spatial conformation.

“Binding affinity” generally refers to the strength of the sum total ofnoncovalent interactions between a single binding site of a molecule(e.g., an antibody) and its binding partner (e.g., an antigen). Unlessindicated otherwise, as used herein, “binding affinity” refers tointrinsic binding affinity which reflects a 1:1 interaction betweenmembers of a binding pair (e.g., antibody and antigen). The affinity ofa molecule X for its partner Y can generally be represented by thedissociation constant (Kd) or the half-maximal effective concentration(EC50). Affinity can be measured by common methods known in the art,including those described herein. Low-affinity antibodies generally bindantigen slowly and tend to dissociate readily, whereas high-affinityantibodies generally bind antigen faster and tend to remain boundlonger. A variety of methods of measuring binding affinity are known inthe art, any of which can be used for purposes of the present invention.Specific illustrative embodiments are described herein.

“Or better” when used herein to refer to binding affinity refers to astronger binding between a molecule and its binding partner. “Or better”when used herein refers to a stronger binding, represented by a smallernumerical Kd value. For example, an antibody which has an affinity foran antigen of “0.6 nM or better,” the antibody's affinity for theantigen is <0.6 nM, i.e., 0.59 nM, 0.58 nM, 0.57 nM etc. or any valueless than 0.6 nM. In one embodiment, the antibody's affinity asdetermined by a Kd will be between about 10⁻³ to about 10⁻¹² M, betweenabout 10⁻⁶ to about 10⁻¹¹ M, between about 10⁻⁶ to about 10⁻¹⁰ M,between about 10⁻⁶ to about 10⁻⁹ M, between about 10⁻⁶ to about 10⁻⁸ M,or between about 10⁻⁶ to about 10⁻⁷ M.

The phrase “substantially similar,” or “substantially the same,” as usedherein, denotes a sufficiently high degree of similarity between twonumeric values (generally one associated with an antibody of theinvention and the other associated with a reference/comparator antibody)such that one of skill in the art would consider the difference betweenthe two values to be of little or no biological and/or statisticalsignificance within the context of the biological characteristicsmeasured by said values (e.g., Kd values). The difference between saidtwo values is less than about 50%, less than about 40%, less than about30%, less than about 20%, or less than about 10% as a function of thevalue for the reference/comparator antibody.

The term “immunoconjugate” or “conjugate” as used herein refers to acompound or a derivative thereof that is linked to a cell binding agent(i.e., an anti-FOLR1 antibody or fragment thereof) and is defined by ageneric formula: A-L-C, wherein C=cytotoxin, L=linker, and A=cellbinding agent or anti-FOLR1 antibody or antibody fragment.Immunoconjugates can also be defined by the generic formula in reverseorder: C-L-A.

A “linker” is any chemical moiety that is capable of linking a compound,usually a drug, such as a maytansinoid, to a cell-binding agent such asan anti-FOLR1 antibody or a fragment thereof in a stable, covalentmanner. Linkers can be susceptible to or be substantially resistant toacid-induced cleavage, light-induced cleavage, peptidase-inducedcleavage, esterase-induced cleavage, and disulfide bond cleavage, atconditions under which the compound or the antibody remains active.Suitable linkers are well known in the art and include, for example,disulfide groups, thioether groups, acid labile groups, photolabilegroups, peptidase labile groups and esterase labile groups. Linkers alsoinclude charged linkers, and hydrophilic forms thereof as describedherein and know in the art.

A polypeptide, antibody, polynucleotide, vector, cell, or compositionwhich is “isolated” is a polypeptide, antibody, polynucleotide, vector,cell, or composition which is in a form not found in nature. Isolatedpolypeptides, antibodies, polynucleotides, vectors, cells orcompositions include those which have been purified to a degree thatthey are no longer in a form in which they are found in nature. In someembodiments, an antibody, polynucleotide, vector, cell, or compositionwhich is isolated is substantially pure.

As used herein, “substantially pure” refers to material which is atleast 50% pure (i.e., free from contaminants), at least 90% pure, atleast 95% pure, at least 98% pure, or at least 99% pure.

The terms “elevated” FOLR1, “increased expression” of FOLR1 and“overexpression” of FOLR1 refer to a sample which contains elevatedlevels of FOLR1 expression. The FOLR1 can be elevated, increased, oroverexpressed as compared to a control value (e.g., expression level ina biological sample, tissue, or cell from a subject without cancer, asample or cancer known to express no or low FOLR1, a normal sample, or acancer that does not have elevated FOLR1 values). For example, a samplewith increased expression can contain an increase of at least 2-fold, atleast 3-fold, or at least 5-fold relative to a control values.

FOLR1 expression can be measured by immunohistochemistry and given astaining intensity score or a staining uniformity score by comparison tocalibrated controls exhibiting defined scores (e.g., an intensity scoreof 3 is given to the test sample if the intensity is comparable to thelevel 3 calibrated control or an intensity of 2 is given to the testsample if the intensity is comparable to the level 2 calibratedcontrol). For example, a score of 1, 2, or 3 (3+), preferably a score of2, or 3 (3+), by immunohistochemistry indicates an increased expressionof FOLR1. A staining uniformity that is heterogeneous or homogeneous isalso indicative of FOLR1 expression. The staining intensity and staininguniformity scores can be used alone or in combination (e.g., 2 homo, 2hetero, 3 homo, 3 hetero, etc.). See Table 11. In another example, anincrease in FOLR1 expression can be determined by detection of anincrease of at least 2-fold, at least 3-fold, or at least 5-foldrelative to control values (e.g., expression level in a tissue or cellfrom a subject without cancer or with a cancer that does not haveelevated FOLR1 values).

A “reference sample” can be used to correlate and compare the resultsobtained in the methods of the invention from a test sample. Referencesamples can be cells (e.g., cell lines, cell pellets) or tissue. TheFOLR1 levels in the “reference sample” can be an absolute or relativeamount, a range of amount, a minimum and/or maximum amount, a meanamount, and/or a median amount of FOLR1. A “reference sample” can alsoserve as a baseline of FOLR1 expression to which the test sample iscompared. The “reference sample” can include a prior sample or baselinesample from the same patient, a normal reference with a known level ofFOLR1 expression, or a reference from a relevant patient population witha known level of FOLR1 expression. FOLR1 levels can also be expressed asvalues in a standard curve. A standard curve is a quantitative method ofplotting assay data to determine the concentration of FOLR1 in a sample.In one embodiment, a reference sample is an antigen standard comprisingpurified FOLR1 or FOLR1-Fc. The diagnostic methods of the invention caninvolve a comparison between expression levels of FOLR1 in a test sampleand a “reference value.” In some embodiments, the reference value is theexpression level of the FOLR1 in a reference sample. A reference valuecan be a predetermined value and can also be determined from referencesamples (e.g., control biological samples or reference samples) testedin parallel with the test samples. A reference value can be a singlecut-off value, such as a median or mean or a range of values, such as aconfidence interval. Reference values can be established for varioussubgroups of individuals, such as individuals predisposed to cancer,individuals having early or late stage cancer, male and/or femaleindividuals, or individuals undergoing cancer therapy. Examples ofnormal reference samples or values and positive reference samples orvalues are described herein and are also described in Examples 1 andExamples 8-10 of WO 2012/135675 herein incorporated by reference.

In some embodiments, the reference sample is a sample from a healthytissue, in particular a corresponding tissue which is not affected bycancer or a corresponding tissue which is not affected by a cancer thatoverexpresses FOLR1 or a corresponding healthy tissue that is known notto express detectable levels of FOLR. These types of reference samplesare referred to as negative control samples or “normal” referencesamples. In other embodiments, the reference sample is a sample from atumor or healthy tissue that expresses detectable FOLR1. These types ofreference samples are referred to as positive control or positivereference samples. Positive control samples can also be used as acomparative indicator for the type (hetero versus homo) and/or degree(0, 1, 2, 3) of staining intensity, which correlates with the level ofFOLR1 expression. Positive control comparative samples are also referredto as calibrated reference samples. Low or non-FOLR1 expressingreferences are described herein in the Examples and also include allstructures of the esophagus, acinar cells/islets of the pancreas,interalveolar connective tissue of lung, and acinar cells of thesalivary gland. For cell lines, exemplary non-expressors include BxPC3,Panc-1, and ASPC1. Positive FOLR1 references are described herein, forexample, in the Examples and also include ducts of pancreas, respiratoryepithelium of normal lung, and intercalated ducts of the salivary gland.In some embodiments, positive FOLR1 references include ducts of pancreasand intercalated ducts of the salivary gland. For cell lines, exemplaryhigh FOLR1 expressors are described herein, for example, in the Examplesand also include KB, HeLa, 300.19 cells transfected with FOLR1, Igrov-1,and Wish, and exemplary low FOLR1 expressors include Ovcar-3, Caov-3,SW620, T47D, and Skov-3. Another positive high FOLR1 reference is a cellline stably or transiently transfected with FOLR1. Additional positiveand negative samples for FOLR1 are described in Table 13. Appropriatepositive and negative reference levels of FOLR1 for a particular cancercan be determined by measuring levels of FOLR1 in one or moreappropriate subjects, and such reference levels can be tailored tospecific populations of subjects (e.g., a reference level can beage-matched so that comparisons can be made between FOLR1 levels insamples from subjects of a certain age and reference levels for aparticular disease state, phenotype, or lack thereof in a certain agegroup). Such reference levels can also be tailored to specifictechniques that are used to measure levels of FOLR1 in biologicalsamples (e.g., immunoassays, etc.).

As used herein, “immunohistochemistry” refers to histochemical andimmunologic methods used to analyze, for example, cells or tissues.Thus, the terms “immunohistochemistry,” “immunocytochemistry,” and“immunochemistry” are used interchangeably.

The term “primary antibody” herein refers to an antibody that bindsspecifically to the target protein antigen in a sample. A primaryantibody is generally the first antibody used in an ELISA assay or IHCprocedure. In one embodiment, the primary antibody is the only antibodyused in an IHC procedure. The term “secondary antibody” herein refers toan antibody that binds specifically to a primary antibody, therebyforming a bridge or link between the primary antibody and a subsequentreagent, if any. The secondary antibody is generally the second antibodyused in an immunohistochemical procedure.

A “sample” or “biological sample” of the present invention is ofbiological origin, in specific embodiments, such as from eukaryoticorganisms. In some embodiments, the sample is a human sample, but animalsamples may also be used. Non-limiting sources of a sample for use inthe present invention include solid tissue, biopsy aspirates, ascites,fluidic extracts, blood, plasma, serum, spinal fluid, lymph fluid, theexternal sections of the skin, respiratory, intestinal, andgenitourinary tracts, tears, saliva, milk, tumors, organs, cell culturesand/or cell culture constituents, for example. A “cancerous sample” is asample that contains a cancerous cell. The method can be used to examinean aspect of expression of FOLR1 or a state of a sample, including, butnot limited to, comparing different types of cells or tissues, comparingdifferent developmental stages, and detecting or determining thepresence and/or type of disease or abnormality.

For the purposes herein, a “section” of a tissue sample regards a singlepart or piece of a tissue sample, e.g. a thin slice of tissue or cellscut from a tissue sample. It is understood that multiple sections oftissue samples may be taken and subjected to analysis according to thepresent invention. In some cases, the selected portion or section oftissue comprises a homogeneous population of cells. In other cases, theselected portion comprises a region of tissue, e.g. the lumen as anon-limiting example. The selected portion can be as small as one cellor two cells, or could represent many thousands of cells, for example.In most cases, the collection of cells is important, and while theinvention has been described for use in the detection of cellularcomponents, the method may also be used for detecting non-cellularcomponents of an organism (e.g. soluble components in the blood as anon-limiting example).

As used herein, the term “capture reagent” refers to a reagent capableof binding and capturing a target molecule in a sample such that undersuitable condition, the capture reagent-target molecule complex can beseparated from the rest of the sample. In one embodiment, the capturereagent is immobilized. In one embodiment, the capture reagent in asandwich immunoassay is an antibody or a mixture of different antibodiesagainst a target antigen.

As used herein, the term “detectable antibody” refers to an antibodythat is capable of being detected either directly through a labelamplified by a detection means, or indirectly through, e.g., anotherantibody that is labeled. For direct labeling, the antibody is typicallyconjugated to a moiety that is detectable by some means. In oneembodiment, the detectable antibody is a biotinylated antibody.

As used herein, the term “detection means” refers to a moiety ortechnique used to detect the presence of the detectable antibody andincludes detection agents that amplify the immobilized label such aslabel captured onto a microtiter plate. In one embodiment, the detectionmeans is a fluorimetric detection agent such as avidin or streptavidin.

Commonly a “sandwich ELISA” employs the following steps: (1) microtiterplate is coated with a capture antibody; (2) sample is added, and anyantigen present binds to capture antibody; (3) detecting antibody isadded and binds to antigen; (4) enzyme-linked secondary antibody isadded and binds to detecting antibody; and (5) substrate is added and isconverted by enzyme to detectable form.

The word “label” when used herein refers to a detectable compound orcomposition which is conjugated directly or indirectly to the antibodyso as to generate a “labeled” antibody. The label can be detectable byitself (e.g. radioisotope labels or fluorescent labels) or, in the caseof an enzymatic label, can catalyze chemical alteration of a substratecompound or composition which is detectable.

By “correlate” or “correlating” is meant comparing, in any way, theperformance and/or results of a first analysis with the performanceand/or results of a second analysis. For example, one may use theresults of a first analysis in carrying out the second analysis and/orone may use the results of a first analysis to determine whether asecond analysis should be performed and/or one may compare the resultsof a first analysis with the results of a second analysis. In oneembodiment, increased expression of FOLR1 correlates with increasedlikelihood of effectiveness of a FOLR1-targeting anti-cancer therapy.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals in which a population of cells arecharacterized by unregulated cell growth. Examples of cancer include,but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, andleukemia. More particular examples of such cancers include cancers ofendothelial, mesenchymal, or epithelial origin, such as lung cancer(e.g., squamous cell cancer, small-cell lung cancer, non-small cell lungcancer, adenocarcinoma of the lung, mesothelioma, and squamous carcinomaof the lung), cancer of the peritoneum (e.g., primary peritoneal),hepatocellular cancer, gastrointestinal cancer, pancreatic cancer,glioblastoma, cervical cancer, ovarian cancer (serous or endometrioid),liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer,colorectal cancer, endometrioid (e.g., endometrial adenocarcinoma) oruterine carcinoma, salivary gland carcinoma, kidney cancer, livercancer, prostate cancer, vulval cancer, thyroid cancer, hepaticcarcinoma, brain cancer (e.g. glioblastoma, tumors of the choroidplexus) and various types of head and neck cancers, and also tumors ofblood vessels and fallopian tubes. Cancers also encompass cancers whichcontain cells having elevated FOLR1 expression levels. SuchFOLR1-elevated cancers include, but are not limited to, ovarian,non-small cell lung cancer (adenocarcinoma), uterine, endometrial,pancreatic, renal, lung, and breast cancer.

“Tumor” and “neoplasm” refer to any mass of tissue that result fromexcessive cell growth or proliferation, either benign (noncancerous) ormalignant (cancerous) including pre-cancerous lesions.

The terms “cancer cell,” “tumor cell,” and grammatical equivalents referto the total population of cells derived from a tumor or a pre-cancerouslesion, including both non-tumorigenic cells, which comprise the bulk ofthe tumor cell population, and tumorigenic stem cells (cancer stemcells). As used herein, the term “tumor cell” will be modified by theterm “non-tumorigenic” when referring solely to those tumor cellslacking the capacity to renew and differentiate to distinguish thosetumor cells from cancer stem cells.

The term “subject” refers to any animal (e.g., a mammal), including, butnot limited to humans, non-human primates, rodents, and the like, whichis to be the recipient of a particular treatment. Typically, the terms“subject” and “patient” are used interchangeably herein in reference toa human subject.

The term “pharmaceutical formulation” refers to a preparation which isin such form as to permit the biological activity of the activeingredient to be effective, and which contains no additional componentswhich are unacceptably toxic to a subject to which the formulation wouldbe administered. Such formulation can be sterile.

An “effective amount” of an antibody or immunoconjugate as disclosedherein is an amount sufficient to carry out a specifically statedpurpose. An “effective amount” can be determined empirically and in aroutine manner, in relation to the stated purpose.

The term “therapeutically effective amount” refers to an amount of anantibody or other drug effective to “treat” a disease or disorder in asubject or mammal. In the case of cancer, the therapeutically effectiveamount of the drug can reduce the number of cancer cells; reduce thetumor size; inhibit (i.e., slow to some extent and in a certainembodiment, stop) cancer cell infiltration into peripheral organs;inhibit (i.e., slow to some extent and in a certain embodiment, stop)tumor metastasis; inhibit, to some extent, tumor growth; relieve to someextent one or more of the symptoms associated with the cancer; and/orresult in a favorable response such as increased progression-freesurvival (PFS), disease-free survival (DFS), or overall survival (OS),complete response (CR), partial response (PR), or, in some cases, stabledisease (SD), a decrease in progressive disease (PD), a reduced time toprogression (TTP), a decrease in CA125 in the case of ovarian cancer, orany combination thereof. See the definition herein of “treating.” To theextent the drug can prevent growth and/or kill existing cancer cells, itcan be cytostatic and/or cytotoxic. In certain embodiments,identification of increased FOLR1 levels allows for administration ofdecreased amounts of the FOLR1-targeting therapeutic to achieve the sametherapeutic effect as seen with higher dosages. A “prophylacticallyeffective amount” refers to an amount effective, at dosages and forperiods of time necessary, to achieve the desired prophylactic result.Typically but not necessarily, since a prophylactic dose is used insubjects prior to or at an earlier stage of disease, theprophylactically effective amount will be less than the therapeuticallyeffective amount.

The term “respond favorably” generally refers to causing a beneficialstate in a subject. With respect to cancer treatment, the term refers toproviding a therapeutic effect on the subject. Positive therapeuticeffects in cancer can be measured in a number of ways (See, W. A. Weber,J. Nucl. Med. 50: 1S-10S (2009)). For example, tumor growth inhibition,molecular marker expression, serum marker expression, and molecularimaging techniques can all be used to assess therapeutic efficacy of ananti-cancer therapeutic. With respect to tumor growth inhibition,according to NCI standards, a T/C≤42% is the minimum level of anti-tumoractivity. A T/C<10% is considered a high anti-tumor activity level, withT/C (%)=Median tumor volume of the treated/Median tumor volume of thecontrol×100. A favorable response can be assessed, for example, byincreased progression-free survival (PFS), disease-free survival (DFS),or overall survival (OS), complete response (CR), partial response (PR),or, in some cases, stable disease (SD), a decrease in progressivedisease (PD), a reduced time to progression (TTP), a decrease in CA125in the case of ovarian cancer or any combination thereof.

PFS, DFS, and OS can be measured by standards set by the National CancerInstitute and the U.S. Food and Drug Administration for the approval ofnew drugs. See Johnson et al, (2003) J. Clin. Oncol. 21(7):1404-1411.

“Progression free survival” (PFS) refers to the time from enrollment todisease progression or death. PFS is generally measured using theKaplan-Meier method and Response Evaluation Criteria in Solid Tumors(RECIST) 1.1 standards. Generally, progression free survival refers tothe situation wherein a patient remains alive, without the cancergetting worse.

“Time to Tumor Progression” (TTP) is defined as the time from enrollmentto disease progression. TTP is generally measured using the RECIST 1.1criteria.

A “complete response” or “complete remission” or “CR” indicates thedisappearance of all signs of tumor or cancer in response to treatment.This does not always mean the cancer has been cured.

A “partial response” or “PR” refers to a decrease in the size or volumeof one or more tumors or lesions, or in the extent of cancer in thebody, in response to treatment.

“Stable disease” refers to disease without progression or relapse. Instable disease there is neither sufficient tumor shrinkage to qualifyfor partial response nor sufficient tumor increase to qualify asprogressive disease.

“Progressive disease” refers to the appearance of one more new lesionsor tumors and/or the unequivocal progression of existing non-targetlesions. Progressive disease can also refer to a tumor growth of morethan 20 percent since treatment began, either due to an increases inmass or in spread of the tumor.

“Disease free survival” (DFS) refers to the length of time during andafter treatment that the patient remains free of disease.

“Overall Survival” (OS) refers to the time from patient enrollment todeath or censored at the date last know n alive. OS includes aprolongation in life expectancy as compared to naive or untreatedindividuals or patients. Overall survival refers to the situationwherein a patient remains alive for a defined period of time, such asone year, five years, etc., e.g., from the time of diagnosis ortreatment.

A “decrease in CA125 levels” can be assessed according to theGynecologic Cancer Intergroup (GCIG) guidelines. For example, CA125levels can be measured prior to treatment to establish a baseline CA125level. CA125 levels can be measured one or more times during or aftertreatment, and a reduction in the CA125 levels over time as compared tothe baseline level is considered a decrease in CA125 levels.

Terms such as “treating” or “treatment” or “to treat” or “alleviating”or “to alleviate” refer to therapeutic measures that cure, slow down,lessen symptoms of, and/or halt progression of a diagnosed pathologiccondition or disorder. Thus, those in need of treatment include thosealready diagnosed with or suspected of having the disorder. In certainembodiments, a subject is successfully “treated” for cancer according tothe methods of the present invention if the patient shows one or more ofthe following: a reduction in the number of or complete absence ofcancer cells; a reduction in the tumor size; inhibition of or an absenceof cancer cell infiltration into peripheral organs including, forexample, the spread of cancer into soft tissue and bone; inhibition ofor an absence of tumor metastasis; inhibition or an absence of tumorgrowth; relief of one or more symptoms associated with the specificcancer; reduced morbidity and mortality; improvement in quality of life;reduction in tumorigenicity, tumorigenic frequency, or tumorigeniccapacity, of a tumor; reduction in the number or frequency of cancerstem cells in a tumor; differentiation of tumorigenic cells to anon-tumorigenic state; increased progression-free survival (PFS),disease-free survival (DFS), or overall survival (OS), complete response(CR), partial response (PR), stable disease (SD), a decrease inprogressive disease (PD), a reduced time to progression (TTP), adecrease in CA125 in the case of ovarian cancer, or any combinationthereof.

Prophylactic or preventative measures refer to therapeutic measures thatprevent and/or slow the development of a targeted pathologic conditionor disorder. Thus, those in need of prophylactic or preventativemeasures include those prone to have the disorder and those in whom thedisorder is to be prevented.

As used herein, the term “healthcare provider” refers to individuals orinstitutions which directly interact with and administer to livingsubjects, e.g., human patients. Non-limiting examples of healthcareproviders include doctors, nurses, technicians, therapist, pharmacists,counselors, alternative medicine practitioners, medical facilities,doctor's offices, hospitals, emergency rooms, clinics, urgent carecenters, alternative medicine clinics/facilities, and any other entityproviding general and/or specialized treatment, assessment, maintenance,therapy, medication, and/or advice relating to all, or any portion of, apatient's state of health, including but not limited to general medical,specialized medical, surgical, and/or any other type of treatment,assessment, maintenance, therapy, medication and/or advice.

In some aspects, a healthcare provider can administer or instructanother healthcare provider to administer a therapy to treat a cancer.“Administration” of a therapy, as used herein, includes prescribing atherapy to a subject as well as delivering, applying, or giving thetherapy to a subject. A healthcare provider can implement or instructanother healthcare provider or patient to perform the following actions:obtain a sample, process a sample, submit a sample, receive a sample,transfer a sample, analyze or measure a sample, quantify a sample,provide the results obtained after analyzing/measuring/quantifying asample, receive the results obtained afteranalyzing/measuring/quantifying a sample, compare/score the resultsobtained after analyzing/measuring/quantifying one or more samples,provide the comparison/score from one or more samples, obtain thecomparison/score from one or more samples, administer a therapy ortherapeutic agent (e.g., a FOLR1 binding agent), commence theadministration of a therapy, cease the administration of a therapy,continue the administration of a therapy, temporarily interrupt theadministration of a therapy, increase the amount of an administeredtherapeutic agent, decrease the amount of an administered therapeuticagent, continue the administration of an amount of a therapeutic agent,increase the frequency of administration of a therapeutic agent,decrease the frequency of administration of a therapeutic agent,maintain the same dosing frequency on a therapeutic agent, replace atherapy or therapeutic agent by at least another therapy or therapeuticagent, combine a therapy or therapeutic agent with at least anothertherapy or additional therapeutic agent. These actions can be performedby a healthcare provider automatically using a computer-implementedmethod (e.g., via a web service or stand-alone computer system).

“Polynucleotide” or “nucleic acid,” as used interchangeably herein,refer to polymers of nucleotides of any length, and include DNA and RNA.The nucleotides can be deoxyribonucleotides, ribonucleotides, modifiednucleotides or bases, and/or their analogs, or any substrate that can beincorporated into a polymer by DNA or RNA polymerase. A polynucleotidecan comprise modified nucleotides, such as methylated nucleotides andtheir analogs. If present, modification to the nucleotide structure canbe imparted before or after assembly of the polymer. The sequence ofnucleotides can be interrupted by non-nucleotide components. Apolynucleotide can be further modified after polymerization, such as byconjugation with a labeling component. Other types of modificationsinclude, for example, “caps,” substitution of one or more of thenaturally occurring nucleotides with an analog, internucleotidemodifications such as, for example, those with uncharged linkages (e.g.,methyl phosphonates, phosphotriesters, phosphoamidates, cabamates, etc.)and with charged linkages (e.g., phosphorothioates, phosphorodithioates,etc.), those containing pendant moieties, such as, for example, proteins(e.g., nucleases, toxins, antibodies, signal peptides, ply-L-lysine,etc.), those with intercalators (e.g., acridine, psoralen, etc.), thosecontaining chelators (e.g., metals, radioactive metals, boron, oxidativemetals, etc.), those containing alkylators, those with modified linkages(e.g., alpha anomeric nucleic acids, etc.), as well as unmodified formsof the polynucleotide(s). Further, any of the hydroxyl groups ordinarilypresent in the sugars can be replaced, for example, by phosphonategroups, phosphate groups, protected by standard protecting groups, oractivated to prepare additional linkages to additional nucleotides, orcan be conjugated to solid supports. The 5′ and 3′ terminal OH can bephosphorylated or substituted with amines or organic capping groupmoieties of from 1 to 20 carbon atoms. Other hydroxyls can also bederivatized to standard protecting groups. Polynucleotides can alsocontain analogous forms of ribose or deoxyribose sugars that aregenerally known in the art, including, for example, 2′-O-methyl-,2′-O-allyl, 2′-fluoro- or 2′-azido-ribose, carbocyclic sugar analogs,alpha-anomeric sugars, epimeric sugars such as arabinose, xyloses orlyxoses, pyranose sugars, furanose sugars, sedoheptuloses, acyclicanalogs and abasic nucleoside analogs such as methyl riboside. One ormore phosphodiester linkages can be replaced by alternative linkinggroups. These alternative linking groups include, but are not limitedto, embodiments wherein phosphate is replaced by P(O)S (“thioate”),P(S)S (“dithioate”), (O)NR₂ (“amidate”), P(O)R, P(O)OR′, CO or CH₂(“formacetal”), in which each R or R′ is independently H or substitutedor unsubstituted alkyl (1-20 C) optionally containing an ether (—O—)linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl or araldyl. Not alllinkages in a polynucleotide need be identical. The precedingdescription applies to all polynucleotides referred to herein, includingRNA and DNA.

The term “vector” means a construct, which is capable of delivering, andexpressing, one or more gene(s) or sequence(s) of interest in a hostcell. Examples of vectors include, but are not limited to, viralvectors, naked DNA or RNA expression vectors, plasmid, cosmid or phagevectors, DNA or RNA expression vectors associated with cationiccondensing agents, DNA or RNA expression vectors encapsulated inliposomes, and certain eukaryotic cells, such as producer cells.

The terms “polypeptide,” “peptide,” and “protein” are usedinterchangeably herein to refer to polymers of amino acids of anylength. The polymer can be linear or branched, it can comprise modifiedamino acids, and it can be interrupted by non-amino acids. The termsalso encompass an amino acid polymer that has been modified naturally orby intervention; for example, disulfide bond formation, glycosylation,lipidation, acetylation, phosphorylation, or any other manipulation ormodification, such as conjugation with a labeling component. Alsoincluded within the definition are, for example, polypeptides containingone or more analogs of an amino acid (including, for example, unnaturalamino acids, etc.), as well as other modifications known in the art. Itis understood that, because the polypeptides of this invention are basedupon antibodies, in certain embodiments, the polypeptides can occur assingle chains or associated chains. In some embodiments, a polypeptide,peptide, or protein is non-naturally occurring. In some embodiments, apolypeptide, peptide, or protein is purified from other naturallyoccurring components. In some embodiments, the polypeptide, peptide, orprotein is recombinantly produced.

The terms “identical” or percent “identity” in the context of two ormore nucleic acids or polypeptides, refer to two or more sequences orsubsequences that are the same or have a specified percentage ofnucleotides or amino acid residues that are the same, when compared andaligned (introducing gaps, if necessary) for maximum correspondence, notconsidering any conservative amino acid substitutions as part of thesequence identity. The percent identity can be measured using sequencecomparison software or algorithms or by visual inspection. Variousalgorithms and software are known in the art that can be used to obtainalignments of amino acid or nucleotide sequences. One such non-limitingexample of a sequence alignment algorithm is the algorithm described inKarlin et al, 1990, Proc. Natl. Acad. Sci., 87:2264-2268, as modified inKarlin et al., 1993, Proc. Natl. Acad. Sci., 90:5873-5877, andincorporated into the NBLAST and XBLAST programs (Altschul et al., 1991,Nucleic Acids Res., 25:3389-3402). In certain embodiments, Gapped BLASTcan be used as described in Altschul et al., 1997. Nucleic Acids Res.25:3389-3402. BLAST-2, WU-BLAST-2 (Altschul et al., 1996, Methods inEnzvmology, 266:460-480), ALIGN, ALIGN-2 (Genentech, South SanFrancisco, Calif.) or Megalign (DNASTAR) are additional publiclyavailable software programs that can be used to align sequences. Incertain embodiments, the percent identity between two nucleotidesequences is determined using the GAP program in GCG software (e.g.,using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 90and a length weight of 1, 2, 3, 4, 5, or 6). In certain alternativeembodiments, the GAP program in the GCG software package, whichincorporates the algorithm of Needleman and Wunsch (J. Mol. Biol.(48):444-453 (1970)) can be used to determine the percent identitybetween two amino acid sequences (e.g., using either a Blossum 62 matrixor a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and alength weight of 1, 2, 3, 4, 5). Alternatively, in certain embodiments,the percent identity between nucleotide or amino acid sequences isdetermined using the algorithm of Myers and Miller (CABIOS, 4:11-17(1989)). For example, the percent identity can be determined using theALIGN program (version 2.0) and using a PAM120 with residue table, a gaplength penalty of 12 and a gap penalty of 4. Appropriate parameters formaximal alignment by particular alignment software can be determined byone skilled in the art. In certain embodiments, the default parametersof the alignment software are used. In certain embodiments, thepercentage identity “X” of a first amino acid sequence to a secondsequence amino acid is calculated as 100×(Y/Z), where Y is the number ofamino acid residues scored as identical matches in the alignment of thefirst and second sequences (as aligned by visual inspection or aparticular sequence alignment program) and Z is the total number ofresidues in the second sequence. If the length of a first sequence islonger than the second sequence, the percent identity of the firstsequence to the second sequence will be longer than the percent identityof the second sequence to the first sequence.

As a non-limiting example, whether any particular polynucleotide has acertain percentage sequence identity (e.g., is at least 80% identical,at least 85% identical, at least 90% identical, and in some embodiments,at least 95%, 96%, 97%, 98%, or 99% identical) to a reference sequencecan, in certain embodiments, be determined using the Bestfit program(Wisconsin Sequence Analysis Package, Version 8 for Unix, GeneticsComputer Group, University Research Park, 575 Science Drive, Madison,Wis. 53711). Bestfit uses the local homology algorithm of Smith andWaterman, Advances in Applied Mathematics 2: 482 489 (1981), to find thebest segment of homology between two sequences. When using Bestfit orany other sequence alignment program to determine whether a particularsequence is, for instance, 95% identical to a reference sequenceaccording to the present invention, the parameters are set such that thepercentage of identity is calculated over the full length of thereference nucleotide sequence and that gaps in homology of up to 5% ofthe total number of nucleotides in the reference sequence are allowed.

In some embodiments, two nucleic acids or polypeptides of the inventionare substantially identical, meaning they have at least 70%, at least75%, at least 80%, at least 85%, at least 90%, and in some embodimentsat least 95%, 96%, 97%, 98%, 99% nucleotide or amino acid residueidentity, when compared and aligned for maximum correspondence, asmeasured using a sequence comparison algorithm or by visual inspection.In certain embodiments, identity exists over a region of the sequencesthat is at least about 10, about 20, about 40-60 residues in length orany integral value therebetween, or over a longer region than 60-80residues, at least about 90-100 residues, or the sequences aresubstantially identical over the full length of the sequences beingcompared, such as the coding region of a nucleotide sequence forexample.

A “conservative amino acid substitution” is one in which one amino acidresidue is replaced with another amino acid residue having a similarside chain. Families of amino acid residues having similar side chainshave been defined in the art, including basic side chains (e.g., lysine,arginine, histidine), acidic side chains (e.g., aspartic acid, glutamicacid), uncharged polar side chains (e.g., asparagine, glutamine, serine,threonine, tyrosine, cysteine), nonpolar side chains (e.g., glycine,alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). For example, substitution of aphenylalanine for a tyrosine is a conservative substitution. In certainembodiments, conservative substitutions in the sequences of thepolypeptides and antibodies of the invention do not abrogate the bindingof the polypeptide or antibody containing the amino acid sequence, tothe antigen(s), i.e., the FOLR1 to which the polypeptide or antibodybinds. Methods of identifying nucleotide and amino acid conservativesubstitutions which do not eliminate antigen-binding are well-known inthe art (see, e.g., Brummell et al., Biochem. 32: 1180-1 187 (1993);Kobayashi et al. Protein Eng. 12(10):879-884 (1999); and Burks et al.Proc. Natl. Acad. Sci. USA 94: 412-417 (1997)).

As used in the present disclosure and claims, the singular forms “a,”“an,” and “the” include plural forms unless the context clearly dictatesotherwise.

It is understood that wherever embodiments are described herein with thelanguage “comprising,” otherwise analogous embodiments described interms of “consisting of” and/or “consisting essentially of” are alsoprovided.

The term “and/or” as used in a phrase such as “A and/or B” herein isintended to include both “A and B,” “A or B,” “A,” and “B.” Likewise,the term “and/or” as used in a phrase such as “A. B, and/or C” isintended to encompass each of the following embodiments: A, B, and C; A,B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B(alone); and C (alone).

II. FOLR1-Binding Agents

The present invention provides agents that specifically bind humanFOLR1. These agents are referred to herein as “FOLR1-binding agents.” Incertain embodiments, the FOLR1 binding agents are antibodies,immunoconjugates or polypeptides. The amino acid and nucleotidesequences for human FOLR1 are known in the art and are also providedherein as represented by SEQ ID NO: 1 and SEQ ID NO:2.

Human Folate Receptor 1: (SEQ ID NO: 1)MAQRMITQLLLLLVWVAVVGEAQTRIAWARTELLNVCMNAKHHKEKPGPEDKLHEQCRPWRKNACCSTNTSQEAHKDVSYLYRFNWNHCGEMAPACKRHFIQDTCLYECSPNLGPWIQQVDQSWRKERVLNVPLCKEDCEQWWEDCRTSYTCKSNWHKGWNWTSGFNKCAVGAACQPFHFYFPTPTVLCNEIWTHSYKVSNYSRGSGRCIQMWFDPAQGNPNEEVARFYAAAMSGAGPWAAWPFLLSLAL MLLWLLSHuman Folate Receptor 1 Nucleic Acid Sequence: (SEQ ID NO: 2)atggctcagcggatgacaacacagctgctgctccttctagtgtgggtggctgtagtaggggaggctcagacaaggattgcatgggccaggactgagcttctcaatgtctgcatgaacgccaagcaccacaaggaaaagccaggccccgaggacaagttgcatgagcagtgtcgaccctggaggaagaatgcctgctgttctaccaacaccagccaggaagcccataaggatgtttcctacctatatagattcaactggaaccactgtggagagatggcacctgcctgcaaacggcatttcatccaggacacctgcctctacgagtgctcccccaacttggggccctggatccagcaggtggatcagagctggcgcaaagagcgggtactgaacgtgcccctgtgcaaagaggactgtgagcaatggtgggaagattgtcgcacctcctacacctgcaagagcaactggcacaagggctggaactggacttcagggtttaacaagtgcgcagtgggagctgcctgccaacctttccatttctacttccccacacccactgttctgtgcaatgaaatctggactcactcctacaaggtcagcaactacagccgagggagtggccgctgcatccagatatggttcgacccagcccagggcaaccccaatgaggaggtggcgaggttctatgctgcagccatgagtggggctgggccctgggcagcctggcctttcctgcttagcctggccctaatgctgctgtggctgctcagc 

Thus, in some embodiments, the FOLR1 binding agents can bind to anepitope of SEQ ID NO:1.

In some embodiments, an anti-FOLR1 antibody can specifically binds to anepitope of FOLR1 (SEQ ID NO:1), wherein epitope comprises anN-glycosylated amino acid. Such antibodies will therefore bind to FOLR1when it is glycosylated and will not bind to FOLR1 when it is notglycosylated. In other words, the binding of these antibodies isglycol-dependent. These antibodies are advantageous in that they can beused to distinguish between glycosylated and non-glycosylated forms ofFOLR1. Given that glycosylation can be required for membranelocalization, the antibodies can advantageously be used for membranespecific staining.

In some embodiments, the anti-FOLR1 antibody can specifically bind to anepitope of FOLR1 comprising N-glycosylated amino acid 69 of FOLR1. Insome embodiments, the anti-FOLR1 antibody can specifically bind to anepitope of FOLR1 comprising N-glycosylated amino acid 161 of FOLR1. Insome embodiments, the anti-FOLR1 antibody can specifically bind to anepitope of FOLR1 comprising N-glycosylated amino acid 201 of FOLR1.

In certain embodiments, the anti-FOLR1 antibody is the antibody producedby the hybridoma deposited with the American Type Culture Collection(ATCC), located at 10801 University Boulevard, Manassas, Va. 20110 onApr. 16, 2013 under the terms of the Budapest Treaty and having ATCCdeposit no. PTA-120196 (“FOLR1-9.20,” also referred to as “IMGN353.9-20,” “353.9-20,” or “9.20”). In certain embodiments, theanti-FOLR1 antibody is the antibody produced by the hybridoma depositedwith the ATCC on Apr. 16, 2013 and having ATCC deposit no. PTA-120197(“FOLR1-2.1,” also referred to as “IMGN 353.2-1,” “353.2-1,” “2.1,” or“muFRIHC2-1”).

The FOLR1-binding agents include FOLR1-binding agents that comprise theheavy and light chain CDR sequences of (i) muFRIHC2-1, which is alsoknown as “FOLR1-2.1,” “IMGN 353.2-1,” “353.2-1,” or “2.1”, (ii)muFRIHC5-7, which is also known as “IMGN 353.5-7.” “353.5-7” or “5.7,”(iii) “muFRIHC9-20.” which is also known as “FOLR1-9.20,” “IMGN353.9-20,” “353.9-20,” or “9.20,” (iv) resurfaced huFRIHC2-1 version 1.0or 1.01, or (v) CDR grafted huFRIHC2-1 version 1.0 or 1.01, which areprovided in Tables 1 and 2 below. The FOLR1-binding agents also includeFOLR1-binding agents that comprise the heavy and light chain CDRsequences of the composite CDRs provided in Tables 1 and 2 below.

TABLE 1 Variable heavy chain CDR amino acid sequences Antibody VH-CDR1VH-CDR2 VH-CDR3 muFRIHC2-1 NSYIH (SEQ WIYPESLNTQYNEKFKA RGIYYYSPYALDH(2.1″) ID NO: 3) (SEQ ID NO: 4) (SEQ ID NO: 5) muFRIHC5-7 NYYIHWIYPGSFNVEYNEKFKA RGIYFYSPYALDY (″5.7″) (SEQ ID NO: 9) (SEQ ID NO: 10)(SEQ ID NO: 11) muFRIHC9-20 NYYIH WIYPENVNVRYNDKFKA RGIYYYSPYAMDY(″9.20″) (SEQ ID (SEQ ID NO: 16) (SEQ ID NO: 17) NO: 15) CompositeN(Y/S)YIH WIYP(G/E)(S/N)(F/V/L)N RGIY(F/Y)YSPYA (SEQ ID(V/T)(E/R/Q)YN(E/D)KFKA (L/M)D(Y/H)(SEQ NO: 21) (SEQ ID NO: 22)ID NO: 23)

TABLE 2 Variable light chain CDR amino acid sequences Antibody VL-CDR1VL-CDR2 VL-CDR3 muFRIHC2-1 KSSKSLLNSDGFTYLD LVSNHFS (SEQ ID FQSNYLPLT(″2.1″) (SEQ ID NO: 6) NO:7 ) (SEQ ID NO: 8) muFRIHC5-7 KSTESLLNSDGFTYLDLVSNHFS (SEQ ID FQSNYLPLT (″5.7″) (SEQ ID NO: 12) NO: 13)(SEQ ID NO: 14) muFRIHC9-20 KSTKSLLNSDGFTYLD LVSNHFS (SEQ ID FQSNYLPLT(″9.20″) (SEQ ID NO: 18) NO: 19) (SEQ ID NO: 20) CompositeKS(T/S)(K/E)SLLNSDGFTY LVSNHFS(SEQ ID FQSNYLPLT LD (SEQ ID NO:24)NO: 25) (SEQ ID NO: 26)

The FOLR1 binding molecules can be antibodies or antigen bindingfragments that specifically bind to FOLR1 that comprise the CDRs ofantibody 2.1 (i.e., SEQ ID NOs: 3-8), 5.7 (i.e., SEQ ID NOs: 9-14), or9.20 (i.e., SEQ ID NOs: 15-20), with up to four (i.e., 0, 1, 2, 3, or 4)conservative amino acid substitutions per CDR. The FOLR1 bindingmolecules can be antibodies or antigen-binding fragments thatspecifically bind to FOLR1 that comprise the CDRs of the compositesequence shown above (i.e., SEQ ID NOs: 21-26), with up to four (i.e.,0, 1, 2, 3, or 4) conservative amino acid substitutions per CDR.

The FOLR1 binding molecules can be antibodies or antigen-bindingfragments that specifically bind to FOLR1 that comprise the CDRs ofantibody produced by the hybridoma of ATCC deposit no. PTA-120196 orPTA-120197.

Polypeptides can comprise one of the individual variable light chains orvariable heavy chains described herein. Antibodies and polypeptides canalso comprise both a variable light chain and a variable heavy chain.The variable light chain and variable heavy chain sequences of murineantibodies 2.1, 5.7, and 9.20 and humanized 2.1 are provided in Tables 3and 4 below.

TABLE 3 Variable heavy chain amino acid sequences AntibodyVH Amino Acid Sequence (SEQ ID NO) muFRIHC2-1QVQLQQSGPELVKPGASVRISCKASGYTFTNSYIHWVKKRPGQGL (″2.1″)EWIGWIYPESLNTQYNEKFKAKATLTADKSSSTSYMQLSSLTSEDSAVYFCARRGIYYYSPYALDHWGQGASVTVSS (SEQ ID NO: 27) muFRIHC5-7QVQLQQSGPEVVKPGASVRISCKASGYTFTNYYIHWVKQRPGQGL (″5.7″)EWIGWIYPGSFNVEYNEKFKAKATLTADKSSSTVYMQLSSLTSEDSAVYFCARRGIYFYSPYALDYWGQGASVTVSS (SEQ ID NO: 29) muFRIHC9-20QVQLQQSGPDLVKPGASVRISCKASGFTFTNYYIHWVKQRPGQGL (″9.20″)EWIGWIYPENVNVRYNDKFKAKATLTADKSSSTAYMQLSSLTSEDSAVYFCARRGIYYYSPYAMDYWGQGASVTVSS (SEQ ID NO: 31) huFRIHC2-1QVQLVQSGAEVVKPGASVKISCKASGYTFTNSYIHWVKKRPGQGL (resurfaced)EWIGWIYPESLNTQYNQKFQGKATLTADKSSSTSYMQLSSLTSEDSAVYFCARRGIYYYSPYALDHWGQGASVTVSS (SEQ ID NO: 62) huFRIHC2-1QVQLVQSGAEVKKPGASVKVSCKASGYTFTNSYIHWVRQAPGQG (grafted)LEWMGWIYPESLNTQYNEKFKARVTMTRDTSISTAYMELSRLRSDDTAVYYCARRGIYYYSPYALDHWGQGTLVTVSSAS (SEQ ID NO: 65)

TABLE 4 Variable light chain amino acid sequences AntibodyVL Amino Acid Sequence (SEQ ID NO) muFRIHC2-1SDVVLTQTPLSLPVNIGDQASISCKSSKSLLNSDGFTYLDWYLQKPG (″2.1″)QSPQLLIYLVSNHFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQSNYLPLTFGGGTKLEIKR (SEQ ID NO: 28) muFRIHC5-7SDVVLTQTPLSLPVNIGDQASISCKSTESLLNSDGFTYLDWYLQKPG (″5.7″)QSPQLLIYLVSNHFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQSNYLPLTFGGGTKLEVKR (SEQ ID NO: 30) muFRIHC9-20SDVVLTQTPLSLPVNLGDQASISCKSTKSLLNSDGFTYLDWYLQKP (″9.20″)GQSPQLLIYLVSNHFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQSNYLPLTFGGGTKLEIKR (SEQ ID NO: 32) huFRIFIC2-1 v.DVVLTQSPLSLPVNLGQPASISCRSSRSLLNSDGFTYLDWYLQKPGQ 1.0 (resurfaced)SPRLLIYLVSNHFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQSNYLPLTFGQGTKLEIKR (SEQ ID NO: 63) huFRIHC2-1 v.DVVLTQSPLSLPVNLGQPASISCKSSKSLLNSDGFTYLDWYLQKPG 1.01 (resurfaced)QSPRLLIYLVSNHFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQSNYLPLTFGQGTKLEIKR (SEQ ID NO: 64) huFRIHC2-1 v.DIVMTQTPLSLSVTPGQPASISCRSSRSLLNSDGFTYLDWYLQKPGQ 1.0 (ratted)SPQLLIYLVSNHFSGVPDRFSGSGSGTDFTLIKISRVEAEDVGVYYCFQSNYLPLTFGQGTKLEIK (SEQ ID NO: 66) huFRIHC2-1 v.DIVMTQTPLSLSVTPGQPASISCKSSKSLLNSDGFTYLDWYLQKPGQ 1.01 (grafted)SPQLLIYLVSNHFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQSNYLPLTFGQGTKLEIK (SEQ ID NO: 67)

Also provided are polypeptides that comprise: (a) a polypeptide havingat least about 90% sequence identity to SEQ ID NOs:27, 29, 31, 62, or65; and/or (b) a polypeptide having at least about 90% sequence identityto SEQ ID NOs:28, 30, 32, 63, 64, 66, or 67. In certain embodiments, thepolypeptide comprises a polypeptide having at least about 950/%, atleast about 96%, at least about 97%, at least about 98%, or at leastabout 99% sequence identity to SEQ ID NOs:27-32 or 62-67. Thus, incertain embodiments, the polypeptide comprises (a) a polypeptide havingat least about 95% sequence identity to SEQ ID NOs:27, 29, 31, 62, or 65and/or (b) a polypeptide having at least about 95% sequence identity toSEQ ID NOs:28, 30, 32, 63, 64, 66, or 67. In certain embodiments, thepolypeptide comprises (a) a polypeptide having the amino acid sequenceof SEQ ID NOs:27, 29, 31, 62, or 65; and/or (b) a polypeptide having theamino acid sequence of SEQ ID NOs:28, 30, 32, 63, 64, 66, or 67. Incertain embodiments, the polypeptide is an antibody and/or thepolypeptide specifically binds FOLR1. In certain embodiments, thepolypeptide is a murine, chimeric, or humanized antibody thatspecifically binds FOLR1. In certain embodiments, the polypeptide havinga certain percentage of sequence identity to SEQ ID NOs:27-32 or 62-67differs from SEQ ID NOs:27-32 or 62-67 by conservative amino acidsubstitutions only.

Also provided are polypeptides comprising a variable light chain that isat least about 85%, at least about 90%, at least about 95%, or at leastabout 99%, or is identical to the variable light chain sequence of theantibody produced by the hybridoma having ATCC deposit no. PTA-120196 orPTA-120197.

Also provided are polypeptides comprising a variable heavy chain that isat least about 85%, at least about 90%, at least about 95%, or at leastabout 99%, or is identical to the variable heavy chain sequence of theantibody produced by the hybridoma having ATCC deposit no. PTA-120196 orPTA-120197.

Also provided are antibodies and antigen-binding fragments thereofcomprising variable heavy and variable light chain sequences that are atleast about 85%, at least about 90%, at least about 95%, or at leastabout 99%, or identical to the variable heavy and variable light chainsequences of the antibody produced by the hybridoma having ATCC depositno. PTA-120196 or PTA-120197.

In certain embodiments, the antibody or antigen-binding fragment is theantibody produced by the hybridoma having ATCC deposit no. PTA-120196 oran antigen-binding fragment thereof.

In certain embodiments, the antibody or antigen-binding fragment is theantibody produced by the hybridoma having ATCC deposit no. PTA-120197 oran antigen-binding fragment thereof.

Polypeptides can comprise one of the individual light chains or heavychains described herein. Antibodies and polypeptides can also compriseboth a light chain and a heavy chain. The light chain and heavy chainsequences of antibodies 2.1, 5.7, and 9.20 are provided in Tables 5 and6 below.

TABLE 5 Full-length heavy chain amino acid sequences AntibodyFull-Length Heavy Chain Amino Acid Sequence (SEQ ID NO) muFRIHC2-1QVQLQQSGPELVKPGASVRISCKASGYTFTNSYIHWVKKRPGQGLE (″2.1″)WIGWIYPESLNTQYNEKFKAKATLTADKSSSTSYMQLSSLTSEDSAVYFCARRGIYYYSPYALDHWGQGASVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLESDLYTLSSSVTVPSSMRPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMNTNGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK (SEQ ID NO:  33) muFRIHC8-7QVQLQQSGPEVVKPGASVRISCKASGYTFTNYYIHWVKQRPGGLE (″5.7″)WIGWTYPGSFNVEYNEKFKAKATLTADKSSSTVYMQLSSLTSEDSAVYFCARRGIYFYSPYALDYWGQGASVTVSSAKTTPPSVYPLPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLESDLYTLSSSVTVPSSMRPSETVTCNVAHPASSTKVDKKIVPRDCGCKCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMNTNGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK (SEQ ID NO: 35) muFRIHC9-20QVQLQQSGPDLVKPGASVRISCKASGFTFTNYYIHWVKQRPGQGLE (″9.20″)WIGWIYPENVNVRYNDKFKAKATLTADKSSSTAYMQLSSLTSEDSAVYFCARRGIYYYSPYAMDYWGQGASVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLESLYTLSSSVTVPSSMRPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMNTNGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK (SEQ ID NO: 37) muhuMov19QVQLVQSGAEVVKPGASVKISCKASGYTFTGYFMNWVKQSPGQSLEWIGRIHPYDGDTFYNQKFQGKATLTVDKSSNTAHMELLSLTSEDFAVYYCTRYDGSRAMDYWGQGTTVTVSSASTKGPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLESDLYTLSSSVTVPSSMRPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMNTNGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK (SEQ ID NO: 68)

TABLE 6 Full-length light Chain amino acid sequences AntibodyFull-length Light Chain Amino Acid Sequence (SEQ ID NO) muFRIHC2-1SDVVLTQTPLSLPVNIGDQASISCKSSKSLLNSDGFTYLDWYLQKPG (″2.1″)QSPQLLIYLVSNHFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQSNYLPLTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC (SEQ ID NO: 34) muFRIHC5-7SDVVLTQTPLSLPVNIGDQASISCKSTESLLNSDGFTYLDWYLQKPG (″5.7″)QSPQLLIYLVSNHFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQSNYLPLTFGGGTKLEVKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC (SEQ ID NO: 36) muFRIHC9-20SDVVLTQTPLSLPVNLGDQASISCKSTKSLLNSDGFTYLDWYLQKP (″9.20″)GQSPQLLIYLVSNHFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQSNYLPLTFGGGTKLEIKRADAAPTVSIFPRSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC (SEQ ID NO: 38) muhuMov19DIVLTQSPLSLAVSLGQPAIISCKASQSVSFAGTSLMHWYHQKPGQQPRLLIYRASNLEAGVPDRFSGSGSKTDFTLTISPVEAEDATYYCQQSREYPYTFGGGTKLEIKRTDAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC (SEQ ID NO:69)

Also provided are polypeptides that comprise: (a) a polypeptide havingat least about 90% sequence identity to SEQ ID NOs:33, 35, or 37; and/or(b) a polypeptide having at least about 90% sequence identity to SEQ IDNOs:34, 36, or 38. In certain embodiments, the polypeptide comprises apolypeptide having at least about 95%, at least about 96%, at leastabout 97%, at least about 98%, or at least about 99% sequence identityto SEQ ID NOs:33-38. Thus, in certain embodiments, the polypeptidecomprises (a) a polypeptide having at least about 95% sequence identityto SEQ ID NOs:33, 35, or 37, and/or (b) a polypeptide having at leastabout 95% sequence identity to SEQ ID NOs:34, 36, or 38. In certainembodiments, the polypeptide comprises (a) a polypeptide having theamino acid sequence of SEQ ID NOs:33, 35, or 37; and/or (b) apolypeptide having the amino acid sequence of SEQ ID NOs:34, 36, or 38.In certain embodiments, the polypeptide is an antibody and/or thepolypeptide specifically binds FOLR1. In certain embodiments, thepolypeptide is a murine, chimeric, or humanized antibody thatspecifically binds FOLR1. In certain embodiments, the polypeptide havinga certain percentage of sequence identity to SEQ ID NOs:33-38 differsfrom SEQ ID NOs:33-38 by conservative amino acid substitutions only.

Also provided are polypeptides comprising a light chain that is at leastabout 85%, at least about 90%, at least about 95%, or at least about99%, or is identical to the light chain sequence of the antibodyproduced by the hybridoma having ATCC deposit no. PTA-120196 orPTA-120197.

Also provided are polypeptides comprising a heavy chain that is at leastabout 85%, at least about 90%, at least about 95%, or at least about99%, or is identical to the heavy chain sequence of the antibodyproduced by the hybridoma having ATCC deposit no. PTA-120196 orPTA-120197.

Also provided are antibodies and antigen-binding fragments thereofcomprising heavy and light chain sequences that are at least about 85%,at least about 90%, at least about 95%, or at least about 99%, oridentical to the heavy and light chain sequences of the antibodyproduced by the hybridoma having ATCC deposit no. PTA-120196 orPTA-120197.

The affinity or avidity of an antibody for an antigen can be determinedexperimentally using any suitable method well known in the art, e.g.,flow cytometry, enzyme-linked immunoabsorbent assay (ELISA), orradioimmunoassay (RIA), or kinetics (e.g., BIACORE™ analysis). Directbinding assays as well as competitive binding assay formats can bereadily employed. (See, for example, Berzofsky, et al.,“Antibody-Antigen Interactions.” In Fundamental Immunology. Paul, W. E.,Ed., Raven Press: New York, N.Y. (1984); Kuby, Janis Immunology, W. H.Freeman and Company: New York, N.Y. (1992); and methods describedherein. The measured affinity of a particular antibody-antigeninteraction can vary if measured under different conditions (e.g., saltconcentration, pH, temperature). Thus, measurements of affinity andother antigen-binding parameters (e.g., KD or Kd, K_(on), K_(off)) aremade with standardized solutions of antibody and antigen, and astandardized buffer, as known in the art and such as the bufferdescribed herein.

In one aspect, binding assays can be performed using flow cytometry oncells expressing the FOLR1 antigen on the surface. For example,FOLR1-positive cells such as SKOV3 can be incubated with varyingconcentrations of anti-FOLR1 antibodies using 1×10⁵ cells per sample in100 μL FACS buffer (RPMI-1640 medium supplemented with 2% normal goatserum). Then, the cells can be pelleted, washed, and incubated for 1 hwith 100 μL of FITC-conjugated goat-anti-mouse or goat-anti-humanIgG-antibody (such as is obtainable from, for example JacksonLaboratory, 6 μg/mL in FACS buffer). The cells are then pelleted again,washed with FACS buffer and resuspended in 200 μL of PBS containing 1%formaldehyde. Samples can be acquired, for example, using a FACSCaliburflow cytometer with the HTS multiwell sampler and analyzed usingCellQuest Pro (all from BD Biosciences, San Diego, US). For each samplethe mean fluorescence intensity for FL1 (MFI) can be exported andplotted against the antibody concentration in a semi-log plot togenerate a binding curve. A sigmoidal dose-response curve is fitted forbinding curves and EC50 values are calculated using programs such asGraphPad Prism v4 with default parameters (GraphPad software, San Diego,Calif.). EC50 values can be used as a measure for the apparentdissociation constant “Kd” or “KD” for each antibody.

Monoclonal antibodies can be prepared using hybridoma methods, such asthose described by Kohler and Milstein (1975) Nature 256:495. Using thehybridoma method, a mouse, hamster, or other appropriate host animal, isimmunized to elicit the production by lymphocytes of antibodies thatwill specifically bind to an immunizing antigen. Lymphocytes can also beimmunized in vitro. Following immunization, the lymphocytes are isolatedand fused with a suitable myeloma cell line using, for example,polyethylene glycol, to form hybridoma cells that can then be selectedaway from unfused lymphocytes and myeloma cells. Hybridomas that producemonoclonal antibodies directed specifically against a chosen antigen asdetermined by immunoprecipitation, immunoblotting, or by an in vitrobinding assay (e.g., radioimmunoassay (RIA); enzyme-linked immunosorbentassay (ELISA)) can then be propagated either in vitro culture usingstandard methods (Goding, Monoclonal Antibodies: Principles andPractice, Academic Press, 1986) or in vivo as ascites tumors in ananimal. The monoclonal antibodies can then be purified from the culturemedium or ascites fluid as described for polyclonal antibodies.

Alternatively monoclonal antibodies can also be made using recombinantDNA methods as described in U.S. Pat. No. 4,816,567. The polynucleotidesencoding a monoclonal antibody are isolated from mature B-cells orhybridoma cells, such as by RT-PCR using oligonucleotide primers thatspecifically amplify, the genes encoding the heavy and light chains ofthe antibody, and their sequence is determined using conventionalprocedures. The isolated polynucleotides encoding the heavy and lightchains are then cloned into suitable expression vectors, which whentransfected into host cells such as E. coli cells, simian COS cells,Chinese hamster ovary (CHO) cells, or myeloma cells that do nototherwise produce immunoglobulin protein, monoclonal antibodies aregenerated by the host cells. Also, recombinant monoclonal antibodies orfragments thereof of the desired species can be isolated from phagedisplay libraries expressing CDRs of the desired species as described(McCafferty et al., 1990, Nature, 348:552-554; Clackson et al., 1991,Nature, 352:624-628; and Marks et al., 1991, J. Mol. Biol.,222:581-597).

The polynucleotide(s) encoding a monoclonal antibody can further bemodified in a number of different manners using recombinant DNAtechnology to generate alternative antibodies. In some embodiments, theconstant domains of the light and heavy chains of, for example, a mousemonoclonal antibody can be substituted 1) for those regions of, forexample, a human antibody to generate a chimeric antibody or 2) for anon-immunoglobulin polypeptide to generate a fusion antibody. In someembodiments, the constant regions are truncated or removed to generatethe desired antibody fragment of a monoclonal antibody. Site-directed orhigh-density mutagenesis of the variable region can be used to optimizespecificity, affinity, etc. of a monoclonal antibody.

In some embodiments, the monoclonal antibody against the human FOLR1 isa humanized antibody. In certain embodiments, such antibodies are usedtherapeutically to reduce antigenicity and HAMA (human anti-mouseantibody) responses when administered to a human subject.

Methods for engineering, humanizing or resurfacing non-human or humanantibodies can also be used and are well known in the art. A humanized,resurfaced or similarly engineered antibody can have one or more aminoacid residues from a source that is non-human. e.g., but not limited to,mouse, rat, rabbit, non-human primate or other mammal. These non-humanamino acid residues are replaced by residues that are often referred toas “import” residues, which are typically taken from an “import”variable, constant or other domain of a known human sequence.

Such imported sequences can be used to reduce immunogenicity or reduce,enhance or modify binding, affinity, on-rate, off-rate, avidity,specificity, half-life, or any other suitable characteristic, as knownin the art. In general, the CDR residues are directly and mostsubstantially involved in influencing FOLR1 binding. Accordingly, partor all of the non-human or human CDR sequences are maintained while thenon-human sequences of the variable and constant regions can be replacedwith human or other amino acids.

Antibodies can also optionally be humanized, resurfaced, engineered orhuman antibodies engineered with retention of high affinity for theantigen FOLR1 and other favorable biological properties. To achieve thisgoal, humanized (or human) or engineered anti-FOLR1 antibodies andresurfaced antibodies can be optionally prepared by a process ofanalysis of the parental sequences and various conceptual humanized andengineered products using three-dimensional models of the parental,engineered, and humanized sequences. Three-dimensional immunoglobulinmodels are commonly available and are familiar to those skilled in theart. Computer programs are available which illustrate and displayprobable three-dimensional conformational structures of selectedcandidate immunoglobulin sequences. Inspection of these displays permitsanalysis of the likely role of the residues in the functioning of thecandidate immunoglobulin sequence, i.e., the analysis of residues thatinfluence the ability of the candidate immunoglobulin to bind itsantigen, such as FOLR1. In this way, framework (FR) residues can beselected and combined from the consensus and import sequences so thatthe desired antibody characteristic, such as increased affinity for thetarget antigen(s), is achieved.

Humanization, resurfacing or engineering of antibodies of the presentinvention can be performed using any known method, such as but notlimited to those described in, Winter (Jones et al., Nature 321:522(1986); Riechmann et al., Nature 332:323 (1988); Verhoeyen et al.,Science 239:1534 (1988)). Sims et al., J. Immunol. 151: 2296 (1993);Chothia and Lesk, J. Mol. Biol. 196:901 (1987), Carter et al., Proc.Natl. Acad. Sci. U.S.A. 89:4285 (1992); Presta et al., J. Immunol.151:2623 (1993), U.S. Pat. Nos. 5,639,641, 5,723,323; 5,976,862;5,824,514; 5,817,483; 5,814,476; 5,763,192; 5,723,323; 5,766,886;5,714,352; 6,204,023; 6,180,370; 5,693,762; 5,530,101; 5,585,089;5,225,539; 4,816,567; PCT/: US98/16280; US96/18978; US91/09630;US91/05939; US94/01234; GB89/01334; GB91/01134; GB92/01755; WO90/14443;WO90/14424; WO90/14430; EP 229246; 7,557,189; 7,538,195; and 7,342,110,each of which is entirely incorporated herein by reference, includingthe references cited therein.

In certain alternative embodiments, the antibody to FOLR1 is a humanantibody. Human antibodies can be directly prepared using varioustechniques known in the art. Immortalized human B lymphocytes immunizedin vitro or isolated from an immunized individual that produce anantibody directed against a target antigen can be generated (See, e.g.,Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p.77 (1985); Boemer et al., 1991, J. Immunol., 147 (1):86-95; and U.S.Pat. No. 5,750,373). Also, the human antibody can be selected from aphage library, where that phage library expresses human antibodies, asdescribed, for example, in Vaughan et al., 1996, Nat. Biotech.,14:309-314, Sheets et al., 1998. Proc. Nat'l. Acad. Sci., 95:6157-6162,Hoogenboom and Winter, 1991. J. Mol. Biol., 227:381, and Marks et al.,1991. J. Mol. Biol., 222:581). Techniques for the generation and use ofantibody phage libraries are also described in U.S. Pat. Nos. 5,969,108,6,172,197, 5,885,793, 6,521,404; 6,544,731; 6,555,313; 6,582,915;6,593.081; 6,300,064; 6,653,068; 6,706,484; and 7,264,963; and Rothe etal., 2007, J. Mol. Bio., doi:10.1016/j.jmb.2007.12.018 (each of which isincorporated by reference in its entirety). Affinity maturationstrategies and chain shuffling strategies (Marks et al., 1992,Bio/Technology 10:779-783, incorporated by reference in its entirety)are known in the art and can be employed to generate high affinity humanantibodies.

Humanized antibodies can also be made in transgenic mice containinghuman immunoglobulin loci that are capable upon immunization ofproducing the full repertoire of human antibodies in the absence ofendogenous immunoglobulin production. This approach is described in U.S.Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and5,661,016.

In certain embodiments are provided an antibody fragment to, forexample, increase tumor penetration. Various techniques are known forthe production of antibody fragments. Traditionally, these fragments arederived via proteolytic digestion of intact antibodies (for exampleMorimoto et al., 1993, Journal of Biochemical and Biophysical Methods24:107-117; Brennan et al., 1985, Science, 229:81). In certainembodiments, antibody fragments are produced recombinantly. Fab, Fv, andscFv antibody fragments can all be expressed in and secreted from E.coli or other host cells, thus allowing the production of large amountsof these fragments. Such antibody fragments can also be isolated fromantibody phage libraries. The antibody fragment can also be linearantibodies as described in U.S. Pat. No. 5,641,870, for example, and canbe monospecific or bispecific. Other techniques for the production ofantibody fragments will be apparent to the skilled practitioner.

For the purposes of the present invention, it should be appreciated thatmodified antibodies can comprise any type of variable region thatprovides for the association of the antibody with the polypeptides of ahuman FOLR1. In this regard, the variable region can comprise or bederived from any type of mammal that can be induced to mount a humoralresponse and generate immunoglobulins against the desired tumorassociated antigen. As such, the variable region of the modifiedantibodies can be, for example, of human, murine, non-human primate(e.g., cynomolgus monkeys, macaques, etc.) or lupine origin. In someembodiments both the variable and constant regions of the modifiedimmunoglobulins are human. In other embodiments the variable regions ofcompatible antibodies (usually derived from a non-human source) can beengineered or specifically tailored to improve the binding properties orreduce the immunogenicity of the molecule. In this respect, variableregions useful in the present invention can be humanized or otherwisealtered through the inclusion of imported amino acid sequences.

In certain embodiments, the variable domains in both the heavy and lightchains are altered by at least partial replacement of one or more CDRsand, if necessary, by partial framework region replacement and sequencechanging. Although the CDRs can be derived from an antibody of the sameclass or even subclass as the antibody from which the framework regionsare derived, it is envisaged that the CDRs will be derived from anantibody of different class and in certain embodiments from an antibodyfrom a different species. It may not be necessary to replace all of theCDRs with the complete CDRs from the donor variable region to transferthe antigen-binding capacity of one variable domain to another. Rather,it may only be necessary to transfer those residues that are necessaryto maintain the activity of the antigen-binding site. Given theexplanations set forth in U.S. Pat. Nos. 5,585,089, 5,693,761 and5,693,762, it will be well within the competence of those skilled in theart, either by carrying out routine experimentation or by trial anderror testing to obtain a functional antibody with reducedimmunogenicity.

Alterations to the variable region notwithstanding, those skilled in theart will appreciate that the modified antibodies of this invention willcomprise antibodies (e.g., full-length antibodies or immunoreactivefragments thereof) in which at least a fraction of one or more of theconstant region domains has been deleted or otherwise altered so as toprovide desired biochemical characteristics such as increased tumorlocalization or reduced serum half-life when compared with an antibodyof approximately the same immunogenicity comprising a native orunaltered constant region. In some embodiments, the constant region ofthe modified antibodies will comprise a human constant region.Modifications to the constant region compatible with this inventioncomprise additions, deletions or substitutions of one or more aminoacids in one or more domains. That is, the modified antibodies disclosedherein can comprise alterations or modifications to one or more of thethree heavy chain constant domains (CH1, CH2 or CH3) and/or to the lightchain constant domain (CL). In some embodiments, modified constantregions wherein one or more domains are partially or entirely deletedare contemplated. In some embodiments, the modified antibodies willcomprise domain deleted constructs or variants wherein the entire CH2domain has been removed (ΔCH2 constructs). In some embodiments, theomitted constant region domain will be replaced by a short amino acidspacer (e.g., 10 residues) that provides some of the molecularflexibility typically imparted by the absent constant region.

It will be noted that in certain embodiments, the modified antibodiescan be engineered to fuse the CH3 domain directly to the hinge region ofthe respective modified antibodies. In other constructs it may bedesirable to provide a peptide spacer between the hinge region and themodified CH2 and/or CH3 domains. For example, compatible constructscould be expressed wherein the CH2 domain has been deleted and theremaining CH3 domain (modified or unmodified) is joined to the hingeregion with a 5-20 amino acid spacer. Such a spacer can be added, forinstance, to ensure that the regulatory elements of the constant domainremain free and accessible or that the hinge region remains flexible.However, it should be noted that amino acid spacers can, in some cases,prove to be immunogenic and elicit an unwanted immune response againstthe construct. Accordingly, in certain embodiments, any spacer added tothe construct will be relatively non-immunogenic, or even omittedaltogether, so as to maintain the desired biochemical qualities of themodified antibodies.

Besides the deletion of whole constant region domains, it will beappreciated that the antibodies of the present invention can be providedby the partial deletion or substitution of a few or even a single aminoacid. For example, the mutation of a single amino acid in selected areasof the CH2 domain may be enough to substantially reduce Fc binding andthereby increase tumor localization. Similarly, it may be desirable tosimply delete that part of one or more constant region domains thatcontrol the effector function (e.g., complement C1Q binding) to bemodulated. Such partial deletions of the constant regions can improveselected characteristics of the antibody (serum half-life) while leavingother desirable functions associated with the subject constant regiondomain intact. Moreover, as alluded to above, the constant regions ofthe disclosed antibodies can be modified through the mutation orsubstitution of one or more amino acids that enhances the profile of theresulting construct. In this respect it may be possible to disrupt theactivity provided by a conserved binding site (e.g., Fc binding) whilesubstantially maintaining the configuration and immunogenic profile ofthe modified antibody. Certain embodiments can comprise the addition ofone or more amino acids to the constant region to enhance desirablecharacteristics such as decreasing or increasing effector function orprovide for more cytotoxin or carbohydrate attachment. In suchembodiments it can be desirable to insert or replicate specificsequences derived from selected constant region domains.

The present invention further embraces variants and equivalents whichare substantially homologous to the chimeric, humanized and humanantibodies, or antibody fragments thereof, set forth herein. These cancontain, for example, conservative substitution mutations, i.e., thesubstitution of one or more amino acids by similar amino acids. Forexample, conservative substitution refers to the substitution of anamino acid with another within the same general class such as, forexample, one acidic amino acid with another acidic amino acid, one basicamino acid with another basic amino acid or one neutral amino acid byanother neutral amino acid. What is intended by a conservative aminoacid substitution is well known in the art.

The polypeptides of the present invention can be recombinantpolypeptides, natural polypeptides, or synthetic polypeptides comprisingan antibody, or fragment thereof, against a human FOLR1. It will berecognized in the art that some amino acid sequences of the inventioncan be varied without significant effect of the structure or function ofthe protein. Thus, the invention further includes variations of thepolypeptides which show substantial activity or which include regions ofan antibody, or fragment thereof, against a human folate receptorprotein. Such mutants include deletions, insertions, inversions,repeats, and type substitutions.

The polypeptides and analogs can be further modified to containadditional chemical moieties not normally part of the protein. Thosederivatized moieties can improve the solubility, the biological halflife or absorption of the protein. The moieties can also reduce oreliminate any desirable side effects of the proteins and the like. Anoverview for those moieties can be found in REMINGTON'S PHARMACEUTICALSCIENCES, 20th ed., Mack Publishing Co., Easton, Pa. (2000).

The isolated polypeptides described herein can be produced by anysuitable method known in the art. Such methods range from direct proteinsynthetic methods to constructing a DNA sequence encoding isolatedpolypeptide sequences and expressing those sequences in a suitabletransformed host. In some embodiments, a DNA sequence is constructedusing recombinant technology by isolating or synthesizing a DNA sequenceencoding a wild-type protein of interest. Optionally, the sequence canbe mutagenized by site-specific mutagenesis to provide functionalanalogs thereof. See, e.g., Zoeller et al., Proc. Nat'l. Acad. Sci. USA81:5662-5066 (1984) and U.S. Pat. No. 4,588,585.

In some embodiments a DNA sequence encoding a polypeptide of interestwould be constructed by chemical synthesis using an oligonucleotidesynthesizer. Such oligonucleotides can be designed based on the aminoacid sequence of the desired polypeptide and selecting those codons thatare favored in the host cell in which the recombinant polypeptide ofinterest will be produced. Standard methods can be applied to synthesizean isolated polynucleotide sequence encoding an isolated polypeptide ofinterest. For example, a complete amino acid sequence can be used toconstruct a back-translated gene. Further, a DNA oligomer containing anucleotide sequence coding for the particular isolated polypeptide canbe synthesized. For example, several small oligonucleotides coding forportions of the desired polypeptide can be synthesized and then ligated.The individual oligonucleotides typically contain 5′ or 3′ overhangs forcomplementary assembly.

Once assembled (by synthesis, site-directed mutagenesis or anothermethod), the polynucleotide sequences encoding a particular isolatedpolypeptide of interest will be inserted into an expression vector andoperatively linked to an expression control sequence appropriate forexpression of the protein in a desired host. Proper assembly can beconfirmed by nucleotide sequencing, restriction mapping, and expressionof a biologically active polypeptide in a suitable host. As is wellknown in the art, in order to obtain high expression levels of atransfected gene in a host, the gene must be operatively linked totranscriptional and translational expression control sequences that arefunctional in the chosen expression host.

In certain embodiments, recombinant expression vectors are used toamplify and express DNA encoding antibodies, or fragments thereof,against human FOLR1. Recombinant expression vectors are replicable DNAconstructs which have synthetic or cDNA-derived DNA fragments encoding apolypeptide chain of an anti-FOLR1 antibody, or fragment thereof,operatively linked to suitable transcriptional or translationalregulatory elements derived from mammalian, microbial, viral or insectgenes. A transcriptional unit generally comprises an assembly of (1) agenetic element or elements having a regulatory role in gene expression,for example, transcriptional promoters or enhancers, (2) a structural orcoding sequence which is transcribed into mRNA and translated intoprotein, and (3) appropriate transcription and translation initiationand termination sequences. Such regulatory elements can include anoperator sequence to control transcription. The ability to replicate ina host, usually conferred by an origin of replication, and a selectiongene to facilitate recognition of transformants can additionally beincorporated. DNA regions are operatively linked when they arefunctionally related to each other. For example, DNA for a signalpeptide (secretory leader) is operatively linked to DNA for apolypeptide if it is expressed as a precursor which participates in thesecretion of the polypeptide; a promoter is operatively linked to acoding sequence if it controls the transcription of the sequence; or aribosome binding site is operatively linked to a coding sequence if itis positioned so as to permit translation. Structural elements intendedfor use in yeast expression systems include a leader sequence enablingextracellular secretion of translated protein by a host cell.Alternatively, where recombinant protein is expressed without a leaderor transport sequence, it can include an N-terminal methionine residue.This residue can optionally be subsequently cleaved from the expressedrecombinant protein to provide a final product.

The choice of expression control sequence and expression vector willdepend upon the choice of host. A wide variety of expression host/vectorcombinations can be employed. Useful expression vectors for eukaryotichosts, include, for example, vectors comprising expression controlsequences from SV40, bovine papilloma virus, adenovirus andcytomegalovirus. Useful expression vectors for bacterial hosts includeknown bacterial plasmids, such as plasmids from Escherichia coli,including pCR 1, pBR322, pMB9 and their derivatives, wider host rangeplasmids, such as M13 and filamentous single-stranded DNA phages.

Suitable host cells for expression of a FOLR1-binding polypeptide orantibody (or a FOLR1 protein to use as an antigen) include prokaryotes,yeast, insect or higher eukaryotic cells under the control ofappropriate promoters. Prokaryotes include gram negative or grampositive organisms, for example E. coli or bacilli. Higher eukaryoticcells include established cell lines of mammalian origin. Cell-freetranslation systems could also be employed. Appropriate cloning andexpression vectors for use with bacterial, fungal, yeast, and mammaliancellular hosts are described by Pouwels et al. (Cloning Vectors: ALaboratory Manual, Elsevier, N.Y., 1985), the relevant disclosure ofwhich is hereby incorporated by reference. Additional informationregarding methods of protein production, including antibody production,can be found, e.g., in U.S. Patent Publication No. 2008/0187954, U.S.Pat. Nos. 6,413,746 and 6,660,501, and International Patent PublicationNo. WO 04009823, each of which is hereby incorporated by referenceherein in its entirety.

Various mammalian or insect cell culture systems are also advantageouslyemployed to express recombinant protein. Expression of recombinantproteins in mammalian cells can be performed because such proteins aregenerally correctly folded, appropriately modified and completelyfunctional. Examples of suitable mammalian host cell lines includeHEK-293 and HEK-293T, the COS-7 lines of monkey kidney cells, describedby Gluzman (Cell 23:175, 1981), and other cell lines including, forexample, L cells, C127, 3T3, Chinese hamster ovary (CHO), HeLa and BHKcell lines. Mammalian expression vectors can comprise nontranscribedelements such as an origin of replication, a suitable promoter andenhancer linked to the gene to be expressed, and other 5′ or 3′ flankingnontranscribed sequences, and 5′ or 3′ nontranslated sequences, such asnecessary ribosome binding sites, a polyadenylation site, splice donorand acceptor sites, and transcriptional termination sequences.Baculovirus systems for production of heterologous proteins in insectcells are reviewed by Luckow and Summers, Bio/Technology 6:47 (1988).

The proteins produced by a transformed host can be purified according toany suitable method. Such standard methods include chromatography (e.g.,ion exchange, affinity and sizing column chromatography),centrifugation, differential solubility, or by any other standardtechnique for protein purification. Affinity tags such as hexahistidine,maltose binding domain, influenza coat sequence andglutathione-S-transferase can be attached to the protein to allow easypurification by passage over an appropriate affinity column. Isolatedproteins can also be physically characterized using such techniques asproteolysis, nuclear magnetic resonance and x-ray crystallography.

For example, supernatants from systems which secrete recombinant proteininto culture media can be first concentrated using a commerciallyavailable protein concentration filter, for example, an Amicon orMillipore Pellicon ultrafiltration unit. Following the concentrationstep, the concentrate can be applied to a suitable purification matrix.Alternatively, an anion exchange resin can be employed, for example, amatrix or substrate having pendant diethylaminoethyl (DEAE) groups. Thematrices can be acrylamide, agarose, dextran, cellulose or other typescommonly employed in protein purification. Alternatively, a cationexchange step can be employed. Suitable cation exchangers includevarious insoluble matrices comprising sulfopropyl or carboxymethylgroups. Finally, one or more reversed-phase high performance liquidchromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media.e.g., silica gel having pendant methyl or other aliphatic groups, can beemployed to further purify a FOLR1-binding agent. Some or all of theforegoing purification steps, in various combinations, can also beemployed to provide a homogeneous recombinant protein.

Recombinant protein produced in bacterial culture can be isolated, forexample, by initial extraction from cell pellets, followed by one ormore concentration, salting-out, aqueous ion exchange or size exclusionchromatography steps. High performance liquid chromatography (HPLC) canbe employed for final purification steps. Microbial cells employed inexpression of a recombinant protein can be disrupted by any convenientmethod, including freeze-thaw cycling, sonication, mechanicaldisruption, or use of cell lysing agents.

Methods known in the art for purifying antibodies and other proteinsalso include, for example, those described in U.S. Patent PublicationNos. 2008/0312425, 2008/0177048, and 2009/0187005, each of which ishereby incorporated by reference herein in its entirety.

III. Polynucleotides

In certain embodiments, the invention encompasses polynucleotidescomprising polynucleotides that encode a polypeptide that specificallybinds a human FOLR1 receptor or a fragment of such a polypeptide. Forexample, the invention provides a polynucleotide comprising a nucleicacid sequence that encodes an antibody to a human FOLR1 or encodes afragment of such an antibody. The polynucleotides of the invention canbe in the form of RNA or in the form of DNA. DNA includes cDNA, genomicDNA, and synthetic DNA; and can be double-stranded or single-stranded,and if single stranded can be the coding strand or non-coding(anti-sense) strand. In some embodiments, the polynucleotide is a cDNAor a DNA lacking one more endogenous introns.

In some embodiments, a polynucleotide is a non-naturally occurringpolynucleotide. In some embodiments, a polynucleotide is recombinantlyproduced.

In certain embodiments, the polynucleotides are isolated. In certainembodiments, the polynucleotides are substantially pure. In someembodiments, a polynucleotide is purified from natural components.

The invention provides a polynucleotide comprising a polynucleotideencoding a polypeptide comprising a sequence selected from the groupconsisting of SEQ ID NOs:3-38 and 59-67. Also provided is apolynucleotide encoding a polypeptide having at least about 95%, atleast about 96%, at least about 97%, at least about 98%, or at leastabout 99% sequence identity to SEQ ID NOs:3-38 and 59-67.

The invention further provides a polynucleotide comprising a sequenceselected from those shown in Tables 7 and 8 below.

TABLE 7 Variable heavy chain polynucleotide sequences AntibodyVariable Heavy Chain Polynucleotide Sequence (SEQ ID NO) muFRIHC2-1caggtccaactgcagcagtctggacctgagctggtgaagcctggggcttcagtgaggatatcctgcaagg(″2.1″)cttctggctacaccttcacaaactcctatattcactgggtgaaaaagaggcctggacagggacttgagtggattggatggatttatcctgaaagtcttaatactcaatacaatgagaagttcaaggccaaggccacactgactgctgacaagtcctccagcacatcctacatgcagctcagcagtctgacctctgaggactctgcggtctatttctgtgcaagaaggggtatttattactactctccctatgctctggaccactggggtcaaggagcctcagtcaccgtctcctca (SEQ ID NO: 39) muFRIHC5-7caggtccaactgcagcagtctggacctgaggtggtgaagcctggggcttcagtgaggatatcctgcaagg(″5.7″)cttctggctacaccttcacaaactactatatacactgggtgaagcagaggcctggacagggacttgagtggattggatggatttatcctggaagttttaatgttgagtacaatgagaagttcaaggccaaggccacactgactgcagacaaatcctccagcacagtctacatgcaactcagcagcctgacctctgaggactctgcggtctatttctgtgcaagaaggggtatttatttctactctccctatgctttggactactggggtcaaggagcctcagtcaccgtctcctca (SEQ ID NO: 41) muFRIHC9-20caggtccaactgcagcagtctggacctgacctggtgaagcctggggcttcagtgaggatatcctgcaagg(″9.20″)cttctggcttcaccttcacaaactaetatatacactgggtgaagcagaggcctggacagggacttgagtggattggatggatttatcctgaaaatgttaatgttaggtacaatgacaagttcaaggccaaggccacactgactgcagacaaatcctccagcacagcctacatgcagctcagcagcctgacctctgaggactctgcggtctatttctgtgcaagaaggggtatttattactactctccctatgctatggactactggggtcaaggagcctcagtcaccgtctcctca (SEQ ID NO:43) huFRIHC2-1aagcttgccaccATGGGTTGGAGCTGCATTATCCTTTTCCTTGTGGCTA (resurfaced)CAGCTACTGGCGTTCACTCTCAGGTACAATTGGTTCAGTCAGGAGCCGAGGTCGTAAAGCCCGGTGCCAGTGTGAAGATCTCATGCAAGGCAAGCGGTTATACTTTTACAAACTCTTACATTCATTGGGTGAAAAAGCGGCCCGGCCAGGGTCTCGAATGGATCGGCTGGATCTACCCAGAAAGTCTGAACACTCAATACAACCAGAAGTTTCAGGGTAAGGCAACTCTCACTGCCGACAAGAGCTCTAGCACAAGCTATATGCAGTTGTCTAGTTTGACAAGCGAGGATAGCGCAGTTTACTTTTGTGCTCGGCGTGGTATTTATTACTACTCACCTTATGCTCTGGATCACTGGGGACAGGGTGCCTCTGTTACCGTTTCCAGTGCATCCACCaagggcc c (SEQ ID NO: 70)huFRIHC2-1 aagcttgccaccATGGGCTGGAGCTGCATAATCCTCTTCCTCGTAGC (grafted)TACCGCCACTGGGGTGCATTCTCAAGTACAGTTGGTGCAGTCCGGAGCTGAAGTCAAGAAGCCAGGGGCTTCTGTrAAGGTGAGCTGTAAGGCTTCCGGATATACCTTCACAAACAGTTATATCCATTGGGTGAGGCAAGCTCCAGGCCAGGGTCTCGAATGGATGGGATGGATCTACCCCGAGAGTCTGAACACCCAGTACAACGAGAAGTTCAAGGCACGTGTGACCATGACAAGAGACACCTCCATCAGTACAGCCTATATGGAATTGAGCCGTCTCAGAAGTGATGATACAGCAGTGTACTACTGCGCCAGGCGGGGCATCTACTACTACAGCCCATACGCTCTCGACCACTGGGGACAAGGAACACTGGTAACCGTAAGCTCAGCTTCTACAaagggccc (SEQ ID NO: 71)

TABLE 8 Variable light chain polynucleotide sequences AntibodyVariable Light Chain Polynucleotide Sequence (SEQ ID NO) muFRIHC2-1agtgatgttgttctgacccaaactccactctctctgcctgtcaatattggagatcaagcctctatctcttgcaagt(″2.1″)cttctaagagtcttctgaatagtgatggattcacttatttggactggtacctgcagaagccaggccagtctccacagctcctaatatatttggtttctaatcatttttctggagttccagacaggttcagtggcagtgggtcaggaacagatttcacactcaagatcagcagagtggaggctgaggatttgggagtttattattgcttccagagtaactatcttcctctcacgttcggaggggggaccaagctggaaataaaacgg (SEQ ID NO: 40) muFRIHC5-7agtgatgttgttctgacccaaactccactctctctgcctgtcaatattggagatcaagcctctatctcrtgcaagt(″5.7″)ctactgagagtcttctgaatagtgatggattcacttatttggactggtacctgcagaagccaggccagtctccacagctcctaatatatttggtttctaatcatttttctggagttccagacaggttcagtggcagtgggtcaggaacagatttcacactcaagatcagcagagtggaggctgaggatttgggagtttattattgcttccagagtaactatcttcctctcacgttcggaggggggaccaagctggaagtaaaacgg (SEQ ID NO: 42) muFRIHC9-20agtgatgttgttctgacccaaactccactctctctgcctgtcaatcttggagatcaagcctctatctcttgcaagt(″9.20″)ctactaagagtcttctgaatagtgatggattcacttatttggactggtacctgcagaagccaggccagtctccacagctcctaatatatttggtitctaatcatttttctggagttccagacaggttcagtggcagtgggtcaggaacagatttcaccctcaagatcagcagagtggaggctgaggatttgggagtttattattgcltccagagtaactatcttcctctcacgttcggaggggggaccaagctggaaataaaacgg (SEQ ID NO: 44) huFRIHC2-1gaattcgccaccATGGGTTGGTCATGTATAATACTTTTCCTGGTAGC v. 1.0TACTGCTACTGGTGTGCATTCAGATGTGGTGCTGACTCAGTC (resurfaced)ACCCTTGTCTCTCCCAGTCAATCTTGGGCAGCCAGCATCTATCAGCTGCCGAAGCAGCAGGTCTCTCCTGAACTCCGATGGCTTTACTTATCTTGACTGGTATCTCCAGAAGCCAGGACAGTCCCCCCGGCTGCTCATCTACCTGGTTTCTAATCATTTTAGTGGCGTCCCTGACCGCTTCTCTGGGAGTGGAAGTGGGACCGATTTTACACTGAAGATCTCCAGGGTCGAAGCTGAGGACCTTGGGGTTTACTACTGTTTCCAGAGCAACTACCTTCCCTTGACATTCGGCCAGGGAACCAAGCTGGAAATCAAGcgtacg (SEQ ID NO: 72) huFRIHC2-1gaattcgccaccATGGGTTGGTCTTGTATCATTCTGTTCCTGGTCGC v. 1.01CACTGCCACAGGAGTTCACTCAGACGTGGTACTCACACAATC (resurfaced)TCCCCTTTCCCTGCCTGTGAACCTGGGACAGCCAGCCTCAATCAGTTGCAAGAGCTCTAAATCTCTGCTCAATAGCGATGGCTTTACCTACTTGGATTGGTACCTCCAGAAGCCCGGCCAGTCTCCTCGGCTCCTGATTTACCTTGTTTCAAATCACTTTTCAGGCGTGCCTGACCGGTTCTCCGGATCTGGCTCAGGGACAGACTTCACCCTGAAGATCTCCCGCGTCGAGGCAGAGGATCTCGGCGTGTATTACTGTTTCCAAAGTAACTACCTGCCATTGACTTTTGGACAAGGAACTAAACTGGAAATCAAAcgtacg (SEQ ID NO: 73) huFRIHC2-1gaattcgccaccATGGGATGGAGTTGTATTATTCTGTTCTTGGTCGC v. 1.0TACTGCAACAGGCGTTCATTCTGACATCGTAATGACCCAGAC (grafted)ACCTCTGAGTCTGAGTGTCACTCCCGGCCAGCCCGCCTCTATTTCATGTCGTAGCTCTCGCTCCCTGCTCAATTCCGACGGTTTTACCTACTTGGACTGGTATCTTCAGAAACCTGGGCAGAGCCCTCAGCTTCTGATCTATCTGGTGTCCAATCACTTCAGTGGCGTCCCAGACCGATTTTCCGGAAGCGGAAGCGGAACCGACTTTACCCTGAAGATATCCCGCGTCGAAGCAGAGGACGTGGGCGTGTATTATTGCTTTCAAAGCAATTACTTGCCATTGACTTTCGGACAAGGCACAAAACTGGAGATTAAGcgtacg (SEQ ID NO: 74) huFRIHC2-1gaattcgccaccATGGGCTGGTCATGCATCATACTGTTCCTGGTGGC v. 1.01TACAGCAACCGGGGTGCACAGCGATATTGTTATGACACAGAC (grafted)ACCACTGAGTTTGTCAGTGACCCCCGGCCAGCCAGCCTCTATATCCTGCAAGTCCTCAAAAAGTCTCCTGAATAGCGATGGCTTTACCTACCTCGACTGGTATCTTCAGAAGCCCGGTCAAAGCCCTCAGCTGCTGATATATCTGGTGTCTAACCATTTTAGCGGAGTCCCCGACCGCTTTTCAGGCTCCGGCAGTGGCACCGACTTCACCCTTAAGATTTCTCGCGTGGAGGCTGAAGATGTAGGGGTCTACTACTGTTTCCAGTCAAACTACCTGCCACTGACCTTTGGTCAAGGCACTAAGCTCGAAATTAAGcgtacg (SEQ ID NO: 75)

Also provided is a polynucleotide having at least about 95%, at leastabout 96%, at least about 97%, at least about 98%, or at least about 99%sequence identity to any one of SEQ ID NOs:39-44.

Also provided are polynucleotides encoding a variable light chain thatis at least about 85%, at least about 90%, at least about 95%, or atleast about 99%, or is identical to the variable light chain sequence ofthe antibody produced by the hybridoma having ATCC deposit no.PTA-120196 or PTA-120197.

Also provided are polynucleotides comprising a variable lightchain-encoding sequence that is at least about 85%, at least about 90%,at least about 95%, or at least about 99%, or is identical to thevariable light chain-encoding sequence that encodes the variable lightchain of the antibody produced by the hybridoma having ATCC deposit no.PTA-120196 or PTA-120197.

Also provided are polynucleotides encoding a variable heavy chain thatis at least about 85%, at least about 90%, at least about 95%, or atleast about 99%, or is identical to the variable heavy chain sequence ofthe antibody produced by the hybridoma having ATCC deposit no.PTA-120196 or PTA-120197.

Also provided are polynucleotides comprising a variable heavychain-encoding sequence that is at least about 85%, at least about 90°6, at least about 95%, or at least about 99%, or is identical to thevariable heavy chain-encoding sequence that encodes the variable heavychain of the antibody produced by the hybridoma having ATCC deposit no.PTA-120196 or PTA-120197.

In certain embodiments the polynucleotides comprise the coding sequencefor the mature polypeptide fused in the same reading frame to apolynucleotide which aids, for example, in expression and secretion of apolypeptide from a host cell (e.g., a leader sequence which functions asa secretory sequence for controlling transport of a polypeptide from thecell). The polypeptide having a leader sequence is a preprotein and canhave the leader sequence cleaved by the host cell to form the matureform of the polypeptide. The polynucleotides can also encode for aproprotein which is the mature protein plus additional 5′ amino acidresidues. A mature protein having a prosequence is a proprotein and isan inactive form of the protein. Once the prosequence is cleaved anactive mature protein remains.

In certain embodiments the polynucleotides comprise the coding sequencefor the mature polypeptide fused in the same reading frame to a markersequence that allows, for example, for purification of the encodedpolypeptide. For example, the marker sequence can be a hexa-histidinetag supplied by a pQE-9 vector to provide for purification of the maturepolypeptide fused to the marker in the case of a bacterial host, or themarker sequence can be a hemagglutinin (HA) tag derived from theinfluenza hemagglutinin protein when a mammalian host (e.g., COS-7cells) is used.

The present invention further relates to variants of the hereinabovedescribed polynucleotides encoding, for example, fragments, analogs, andderivatives.

The polynucleotide variants can contain alterations in the codingregions, non-coding regions, or both. In some embodiments thepolynucleotide variants contain alterations which produce silentsubstitutions, additions, or deletions, but do not alter the propertiesor activities of the encoded polypeptide. In some embodiments,nucleotide variants are produced by silent substitutions due to thedegeneracy of the genetic code. Polynucleotide variants can be producedfor a variety of reasons, e.g., to optimize codon expression for aparticular host (change codons in the human mRNA to those preferred by abacterial host such as E. coli).

Vectors and cells comprising the polynucleotides described herein arealso provided.

IV. Biological Samples

Biological samples are often fixed with a fixative. Aldehyde fixativessuch as formalin (formaldehyde) and glutaraldehyde are typically used.Tissue samples fixed using other fixation techniques such as alcoholimmersion (Battifora and Kopinski, J. Histochem. Cytochem. (1986)34:1095) are also suitable. The samples used may also be embedded inparaffin. In one embodiment, the samples are both formalin-fixed andparaffin-embedded (FFPE). In another embodiment, the FFPE block ishematoxylin and eosin stained prior to selecting one or more portionsfor analysis in order to select specific area(s) for the FFPE coresample. Methods of preparing tissue blocks from these particulatespecimens have been used in previous IHC studies of various prognosticfactors, and/or is well known to those of skill in the art (see, forexample, Abbondanzo et al., Am J Clin Pathol. 1990 May; 93(5):698-702;Allred et al., Arch Surg. 1990 January; 125(1):107-13).

Briefly, any intact organ or tissue may be cut into fairly small piecesand incubated in various fixatives (e.g. formalin, alcohol, etc.) forvarying periods of time until the tissue is “fixed”. The samples may bevirtually any intact tissue surgically removed from the body. Thesamples may be cut into reasonably small piece(s) that fit on theequipment routinely used in histopathology laboratories. The size of thecut pieces typically ranges from a few millimeters to a few centimeters.The biological sample can also be fluidic extracts, blood, plasma,serum, spinal fluid, lymph fluid, and or splenic preparations.

V. Correlation of FOLR1 Expression and Therapeutic Efficacy

The antibody maytansinoid conjugate (AMC) IMGN853 comprises theFOLR1-binding monoclonal antibody, huMov19 (M9346A), conjugated to themaytansinoid, DM4(N(2′)-deacetyl-N2′-(4-mercapto-4-methyl-1-oxopentyl)-maytansine),attached via the cleavable sulfo-SPDB (N-succinimidyl4-(2-pyridyldithio)-2-sulfobutanoate) linker. The antibody sequences ofIMGN853 (huMov19) are provided below as SEQ ID NOs: 45 and 47, andIMGN853 and huMov19 are described in US Appl. Pub. No. 2012/0009181 (nowU.S. Pat. No. 8,557,966), which is herein incorporated by reference inits entirety.

-huMov19 vHC SEQ ID NO: 45QVQLVQSGAEVVKPGASVKISCKASGYTFTGYFMNWVKQSPGQSLEWIGRIHPYDGDTFYNQKFQGKATLTVDKSSNTAHMELLSLTSEDFAVYYCTRYD GSRAMDYWGQGTTVTVSS-huMov19 vLCv1.00 SEQ ID NO: 46DIVLTQSPLSLAVSLGQPAIISCKASQSVSFAGTSLMHWYHQKPGQQPRLLIYRASNLEAGVPDRFSGSGSKTDFTLNISPVEAEDAATYYCQQSREYPY TFGGGTKLEIKR-huMov19 vLCv1.60 SEQ ID NO: 47DIVLTQSPLSLAVSLGQPAIISCKASQSVSFAGTSLMHWYHQKPGQQPRLLIYRASNLEAGVPDRFSGSGSKTDFTLTISPVEAEDAATYYCQQSREYPY TFGGGTKLEIKR-huMov19 vLC CDR1 SEQ ID NO: 48 KASQSVSFAGTSLMH -huMov19 vLC CDR2SEQ ID NO: 49 RASNLEA -huMov19 vLC CDR3 SEQ ID NO: 50 QQSREYPYT-huMov19 vHC CDR1 SEQ ID NO: 51 GYFMN -huMov19 vHC CDR2-Rabat DefinedSEQ ID NO: 52 RIIIPYDGDTFYNQKFQG -huMov19 vHC CDR2-Abm DefinedSEQ ID NO: 53 RIHPYDGDTF -huMov19 vHC CDR3 SEQ ID NO: 54 YDGSRAMDY-huMov19 HC amino acid sequence SEQ ID NO: 55QVQLVQSGAEVVKPGASVKISCKASGYTFTGYFMNWVKQSPGQSLEWIGRIHPYDGDTFYNQKFQGKATLTVDKSSNTAHMELLSLTSEDFAVYYCTRYDGSRAMDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK -huMov19 LCv1.00SEQ ID NO: 56 DIVLTQSPLSLAVSLGQPAIISCKASQSVSFAGTSLMHWYHQKPGQQPRLLIYRASNLEAGVPDRFSGSGSKTDFTLNISPVEAEDAATYYCQQSREYPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEC-huMov19 LCv1.60 SEQ ID NO: 57DIVLTQSPLSLAVSLGQPAIISCKASQSVSFAGTSLMHWYHQKPGQQPRLLIYRASNLEAGVPDRFSGSGSKTDFTLTISPVEAEDAATYYCQQSREYPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEC-muMov19 vHC CDR2-Rabat Defined SEQ ID NO: 58 RIHPYDGDTFYNQNFKD

IMGN853 is currently in clinical development for various therapeuticindications which include FOLR1 positive ovarian cancer, non-small celllung cancer, endometrioid cancer, renal cancer, and other epithelialmalignancies. Ovarian cancers exhibit the greatest FOLR1 penetrance andare considered the major indications for treatment with IMGN853 (AntonyA C. Ann Rev Nutr 16:501-21 (1996); Yuan Y et al. Hum Pathol40(10):1453-1460 (2009)). Measuring levels of FOLR1 in patient plasmasamples can help identify patient populations more likely to respond toAMC treatment.

In certain embodiments, the invention provides a method for identifyingsubjects that are likely to respond favorably to FOLR1-targetinganti-cancer therapies due to elevated expression levels of FOLR1 beingexpressed in the subject, in particular using antibodies andantigen-binding fragments thereof provided herein that can detect adynamic range of FOLR1 expression levels, e.g., in IHC.

Evaluation of patient samples and correlation to in vivo efficacy usingxenograft models demonstrates the power of the expression analysis forselecting subjects more likely to respond to treatment. IHC provides ascore for FOLR1 expression on tumor cells: 0 (no expression) to 3 (or3+) (very high levels of expression). In vivo data using xenograftmodels demonstrates that samples scoring 2, or 3 (or 3+) for FOLR1expression have an increased likelihood to respond to FOLR1-targetedanti-cancer therapies at clinically-relevant doses of FOLR1immunoconjugates (see e.g., U.S. Provisional Application Nos. 61/823,317and 61/828,586 and International Application No. PCT/US2014/037911, allof which are herein incorporated by reference in their entireties).Thus, identification of individuals having an elevated FOLR1 score wouldhelp identify those individuals who might respond to a clinicallyrelevant dosage. Moreover, expression of more uniform levels of FOLR1provides better correlation with therapeutic benefit. Thus, a homogenousstaining uniformity or a combination of increased staining withheterogenous staining uniformity can indicate increased FOLR1expression. For example, scores of greater than 2 hetero may be used asa patient selection criterion for treatment with a FOLR1 therapeuticagent (see e.g., U.S. Published Application No. 2012/0282175, which isherein incorporated by reference in its entirety).

FOLR1 expression analysis also identifies patients in whom decreasedlevels of a FOLR1-targeting anti-cancer therapy (“low dose therapy”) canbe effective to cause anti-tumor responses. As is appreciated in theart, compounds are generally administered at the smallest dosage thatachieves the desired therapeutic response. This is specificallyimportant for therapeutics that cause clinical side effects. The abilityto recognize those subjects with elevated FOLR1 expression levels allowsfor minimization of the dosage of the FOLR1-targeting therapeutic, thusdecreasing possible side effects, while maintaining therapeuticefficacy.

Accordingly, the antibodies and antigen-binding fragments providedherein are particularly advantageous for use in such methods becausethey are capable of detecting a dynamic range of FOLR1 expressionlevels, e.g., in IHC.

VI. Shed Antigen Assay

Measuring levels of circulating antigen in patient plasma samples (shedantigen) can help identify patient populations more likely to respond totreatment, e.g., antibody maytansinoid conjugate (AMC) treatment. Highlevels of shed antigen have been reported to markedly affect thepharmacokinetics of therapeutic antibodies (Tolcher A. et al. 20thSymposium on Molecular Targets and Cancer Therapeutics; Oct. 21-24,2008; Geneva, Switzerland: EORTC-NCI-AACR, p 163, #514; Baselga J, etal. J Clin Oncol 14:737-744 (1996)). It is likely that shed antigenlevels from patient plasma samples will be variable depending on factorssuch as antigen target, disease indications, and disease course.Currently shed antigen levels in disease indications for the anti-FOLR1immunoconjugate IMGN853 have been insufficiently examined whilecorrelation with solid tumor expression is limited. While elevation ofFOLR1 has been reported in ovarian adenocarcinomas, data suggests thatit is not elevated in other FOLR1+ tumor indications, such as small celllung carcinoma (Mantovani L T, et al. Eur J Cancer 30A(3):363-9 (1994);Basal E, et al. PLoS ONE 4(7): e6292 (2009)). The present method allowsfor detection of the FOLR1 receptor in the presence of high folic acidusing the antibodies and antigen-binding fragments thereof that areprovided herein and are capable of detecting dynamic ranges of shedFOLR1. Previous assays have used Mov19 in the design of the assay. SinceIMGN853 contains Mov19 and in one embodiment is the targeted therapy ofthe invention, it is vital that the method detects FOLR1 in the presenceor absence of Mov19. Previous assays that use Mov19 have competitiveeffects and will detect significantly less or no FOLR1 in patientsreceiving IMGN853 treatment.

In one embodiment, the present method for detecting FOLR1 in humansourced fluid samples uses a traditional sandwich ELISA format. In oneembodiment, the method uses a capture agent (i.e., antibody) to FOLR1attached to a solid support. In one embodiment, the solid support is amicrotiter plate. To this, the sample (ascites fluids, plasma, etc.) isadded without dilution, and is detected by a different detection agent(a different antibody), which does not interfere with the binding of thefirst capture agent. The detection agent is then detected through theuse of a secondary detection agent (biotin/streptavidin, anti-humansecondary mono or polyclonal antibody, etc.) which can bind more thanone time to the first detection agent, thus amplifying the signal ofdetection. The secondary detection agent is then quantified by the useof some other means (e.g., TMB/peroxidase, scintillation counting,fluorescent probes, etc.). Additionally, the assay detects FOLR1 and isnot negatively impacted by the presence of Mov19, IMGN853, other FOLR1family members, or folic acid.

The assays of the present invention include assays both to selectpatients eligible to receive FOLR1-based therapy and assays to monitorpatient response. Assays for response prediction are run before therapyselection, and levels of FOLR1 may impact therapy decisions. Formonitoring patient response, the assay is run at the initiation oftherapy to establish baseline (or predetermined) levels of FOLR1 in thesample. The same sample is then assayed and the levels of FOLR1 comparedto the baseline or predetermined levels. As used herein, the term“predetermined level” refers generally to an assay cutoff value that isused to assess diagnostic results by comparing the assay results againstthe predetermined level, and where the predetermined level already hasbeen linked or associated with various clinical parameters (e.g.,monitoring whether a subject being treated with a drug has achieved anefficacious blood level of the drug, monitoring the response of asubject receiving treatment for cancer with an anti-cancer drug,monitoring the response of a tumor in a subject receiving treatment forsaid tumor, etc.). The predetermined level may be either an absolutevalue or a value normalized by subtracting the value obtained from apatient prior to the initiation of therapy. An example of apredetermined level that can be used is a baseline level obtained fromone or more subjects that may optionally be suffering from one or morediseases or conditions. The comparison (or informational analysis) ofthe level of the assayed biomarker with the baseline or predeterminedlevel can be done by an automated system, such as a software program orintelligence system that is part of, or compatible with, the equipment(e.g., computer platform) on which the assay is carried out.Alternatively, this comparison or informational analysis can be done bya physician. In one embodiment, where the levels remain the same ordecrease, the therapy may be effective and can be continued. Wheresignificant increase over baseline level (or predetermined level)occurs, the patient may not be responding. In another embodiment, anincrease in shed FOLR1 levels may be indicative of increased cell deathand increased release of the shed FOLR1. In this embodiment, an increasein shed FOLR1 is indicative of therapeutic efficacy.

The assays of the present invention can be performed by any proteinassay methods. Protein assay methods useful in the invention are wellknown in the art and include immunoassay methods involving binding of aspecific unlabeled or labeled antibody or protein to the expressedprotein or fragment of FOLR1. Useful immunoassay methods include bothsolution phase assays conducted using any format known in the art, suchas, but not limited to, Biacore, time resolved fluorescence energytransfer (TR-FRET), an ELISA format, (sandwich, forward and reversecompetitive inhibition) or a fluorescence polarization format, and solidphase assays such as immunohistochemistry. The FOLR1 antibodies andantigen-binding fragments thereof provided herein are particularlyuseful for these immunoassay methods because, for example, they are ableto detect a dynamic range of FOLR1.

VII. Circulating Tumor Cell Assays

The anti-FOLR1 antibodies described herein can also be used for thedetection of FOLR1 in a circulating tumor cell assay. Circulating tumorcells (CTCs) are cells that have shed into the vasculature from a tumorand circulate in the bloodstream. CTCs are present in circulation inextremely low numbers. In general, CTCs are enriched from patient bloodor plasma by various techniques known in the art. CTCs can be stainedfor specific markers using methods known in the art including, but notlimited to, flow cytometry-based methods and IHC-based methods. CTCs maybe stained for protein markers unique to the tumor cells, which allowsfor the identification and distinction of CTCs from normal blood cells.CTCs can also be stained for FOLR1 using the antibodies provided hereinincluding but not limited to 2.1, 5.7, and 9.20. CTC analysis can alsoinclude quantitative analysis of the number of CTCs and/or the number ofFOLR1 positive CTCs. The FOLR1 antibodies described herein can be usedto stain the CTCs isolated from a subject having a cancer to measure theFOLR1 present in the CTCs. An increase in FOLR1 expressing CTCs can helpidentify the subject as having a cancer that is likely to respond toFOLR1 based therapy or allow for optimization of a therapeutic regimenwith a FOLR1 antibody or immunoconjugate. CTC FOLR1 quantitation canprovide information on the stage of tumor, response to therapy and/ordisease progression. It can be used as prognostic, predictive orpharmacodimamic biomarker. In addition, staining of CTCs for FOLR1 usingthe antibodies provided herein, can be used as a liquid biopsy eitheralone or in combination with additional tumor marker analysis of solidbiopsy samples.

VIII. Detection

The present invention further provides antibodies against FOLR1,generally of the monoclonal type, that are linked to at least one agentto form a detection antibody conjugate. In order to increase theefficacy of antibody molecules as diagnostic it is conventional to linkor covalently bind or complex at least one desired molecule or moiety.Such a molecule or moiety may be, but is not limited to, at least onereporter molecule. A reporter molecule is defined as any moiety that maybe detected using an assay. Non-limiting examples of reporter moleculesthat have been conjugated to antibodies include enzymes, radiolabels,haptens, fluorescent labels, phosphorescent molecules, chemiluminescentmolecules, chromophores, luminescent molecules, photoaffinity molecules,colored particles and/or ligands, such as biotin.

Certain examples of antibody conjugates are those conjugates in whichthe antibody or antigen-binding fragment thereof provided herein islinked to a detectable label. “Detectable labels” are compounds and/orelements that can be detected due to their specific functionalproperties, and/or chemical characteristics, the use of which allows theantibody or antigen-binding fragment to which they are attached to bedetected, and/or further quantified if desired.

Many appropriate imaging agents are known in the art, as are methods fortheir attachment to antibodies (see, e.g., U.S. Pat. Nos. 5,021,236;4,938,948; and 4,472,509, each incorporated herein by reference). Theimaging moieties used can be paramagnetic ions; radioactive isotopes;fluorochromes; NMR-detectable substances; and/or X-ray imaging, forexample.

Exemplary fluorescent labels contemplated for use as binding agent(e.g., antibody) conjugates include Alexa 350, Alexa 430, Alexa 488,AMCA, BODIPY 630/650, BODIPY 650/665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR,BODIPY-TRX, Cascade Blue, Cy3, Cy5,6-FAM, Dylight 488, FluoresceinIsothiocyanate (FITC), Green fluorescent protein (GFP), HEX, 6-JOE,Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue,Phycoerythrin, REG, Rhodamine Green, Rhodamine Red, tetramethyl rhodamin(TMR) Renographin, ROX, TAMRA, TET, Tetramethylrhodamine, Texas Red, andderivatives of these labels (i.e., halogenated analogues, modified withisothiocynate or other linker for conjugating, etc.), for example. Anexemplary radiolabel is tritium.

Antibody or antigen-binding fragment detection conjugates contemplatedin the present invention include those for use in vitro, where theantibody or fragment is linked to a secondary binding ligand and/or toan enzyme (an enzyme tag) that will generate a colored product uponcontact with a chromogenic substrate. The FOLR1 antibodies andantigen-binding fragments thereof provided herein are particularlyuseful for conjugates methods because, for example, they are able todetect a dynamic range of FOLR1. Examples of suitable enzymes includeurease, alkaline phosphatase, (horseradish) hydrogen peroxidase and/orglucose oxidase. In some embodiments, secondary binding ligands arebiotin and/or avidin and streptavidin compounds. The use of such labelsis well known to those of skill in the art and are described, forexample, in U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345;4,277,437; 4,275,149 and 4,366,241; each incorporated herein byreference.

Molecules containing azido groups may also be used to form covalentbonds to proteins through reactive nitrene intermediates that aregenerated by low intensity ultraviolet light (Potter & Haley, 1983). Inparticular, 2- and 8-azido analogues of purine nucleotides have beenused as site-directed photoprobes to identify nucleotide bindingproteins in crude cell extracts (Owens & Haley, 1987; Atherton et al.,1985). The 2- and 8-azido nucleotides have also been used to mapnucleotide binding domains of purified proteins (Khatoon et al., 1989;King et al., 1989; and Dholakia et al., 1989) and can be used asantibody binding agents.

Several methods are known in the art for the attachment or conjugationof an antibody to its conjugate moiety. Some attachment methods involvethe use of a metal chelate complex employing, for example, an organicchelating agent such a diethylenetriaminepentaacetic acid anhydride(DTPA); ethylenetriaminetetraacetic acid; N-chloro-p-toluenesulfonamide;and/or tetrachloro-3α-6α-diphenylglycouril-3 attached to the bindingagent (e.g., antibody) (U.S. Pat. Nos. 4,472,509 and 4,938,948, eachincorporated herein by reference). Monoclonal antibodies may also bereacted with an enzyme in the presence of a coupling agent such asglutaraldehyde or periodate. Protein binding (e.g., antibody) conjugateswith fluorescein markers are prepared in the presence of these couplingagents or by reaction with an isothiocyanate. In U.S. Pat. No.4,938,948, imaging of breast tumors, for example, is achieved usingmonoclonal antibodies, and the detectable imaging moieties are bound tothe antibody using linkers such as methyl-p-hydroxybenzimidate orN-succinimidyl-3-(4-hydroxyphenyl)propionate.

In other embodiments, derivatization of immunoglobulins by selectivelyintroducing sulfhydryl groups in the Fc region of an immunoglobulinusing reaction conditions that do not alter the antibody combining siteare contemplated. Antibody conjugates produced according to thismethodology are disclosed to exhibit improved longevity, specificity andsensitivity (U.S. Pat. No. 5,196,066, incorporated herein by reference).Site-specific attachment of effector or reporter molecules, wherein thereporter or effector molecule is conjugated to a carbohydrate residue inthe Fc region, have also been disclosed in the literature (O'Shannessyet al., 1987).

In other embodiments of the invention, immunoglobulins are radiolabeledwith nuclides such as tritium. In additional embodiments, nanogoldparticles (such as sizes from about 0.5 nm-40 nm) and/or Quantum Dots(Hayward, Calif.) are employed.

When a sandwich assay format is used, the capture antibody will beunlabeled. The detection antibody will be either directly labeled, ordetected indirectly by addition (after washing off excess detectionantibody) of a molar excess of a second, labeled antibody directedagainst the first antibody.

The label used for the detection antibody is any detectablefunctionality that does not interfere with the binding of the FOLR1antibodies. Examples of suitable labels are those numerous labels knownfor use in immunoassay, including moieties that may be detecteddirectly, such as fluorochrome, chemiluminescent, and radioactivelabels, as well as moieties, such as enzymes, that must be reacted orderivatized to be detected. Examples of such labels include theradioisotopes ³²P, ¹⁴C, ¹²⁵I, ³H, and ¹³¹I, fluorophores such as rareearth chelates or fluorescein and its derivatives, rhodamine and itsderivatives, dansyl, umbelliferone, luciferases, e.g., fireflyluciferase and bacterial luciferase (U.S. Pat. No. 4,737,456),luciferin, 2,3-dihydrophthalazinediones, horseradish peroxidase (HRP),alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme,saccharide oxidases, e.g., glucose oxidase, galactose oxidase, andglucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricaseand xanthine oxidase, coupled with an enzyme that employs hydrogenperoxide to oxidize a dye precursor such as HRP, lactoperoxidase, ormicroperoxidase, biotin/avidin, biotin/streptavidin,biotin/Streptavidin-β-galactosidase with MUG, spin labels, bacteriophagelabels, stable free radicals, and the like. As noted herein, thefluorimetric detection is one example.

Conventional methods are available to bind these labels covalently toproteins or polypeptides. For instance, coupling agents such asdialdehydes, carbodiimides, dimaleimides, bis-imidates, bis-diazotizedbenzidine, and the like may be used to tag the antibodies with theherein-described fluorescent, chemiluminescent, and enzyme labels. See,for example, U.S. Pat. No. 3,940,475 (fluorimetry) and U.S. Pat. No.3,645,090 (enzymes); Hunter et al. Nature 144:945 (1962); David et al.Biochemistry 13:1014-1021 (1974); Pain et al. J. Immunol. Methods40:219-230 (1981); and Nygren J. Histochem. and Cytochem. 30:407-412(1982). In certain embodiments, labels herein are fluorescent toincrease amplification and sensitivity to 8 pg/ml, more preferablybiotin with streptavidin-β-galactosidase and MUG for amplifying thesignal. In certain embodiments, a colorimetric label is used, e.g.,where the detectable antibody is biotinylated and the detection means isavidin or streptavidin-peroxidase and 3,3′,5,5′-tetramethyl benzidine.

The conjugation of such label, including the enzymes, to the antibody isa standard manipulative procedure for one of ordinary skill inimmunoassay techniques. See, for example, O'Sullivan et al. “Methods forthe Preparation of Enzyme-antibody Conjugates for Use in EnzymeImmunoassay,” in Methods in Enzymology, ed. J. J. Langone and H. VanVunakis, Vol. 73 (Academic Press, New York, N.Y., 1981), pp. 147-166.

Following the addition of last labeled antibody, the amount of boundantibody is determined by removing excess unbound labeled antibodythrough washing and then measuring the amount of the attached labelusing a detection method appropriate to the label, and correlating themeasured amount with the amount of shed FOLR1 in the biological sample.For example, in the case of enzymes, the amount of color developed andmeasured will be a direct measurement of the amount of shed FOLR1present. Specifically, if HRP is the label, the color can be detectedusing the substrate 3,3′,5,5′-tetramethyl benzidine at 450 nmabsorbance.

IX. Substrates and Indicators

The use of substrates and indicators is contemplated for detection ofFOLR1.

Horseradish peroxidase (HRP) is an enzyme that first forms a complexwith hydrogen peroxide and then causes it to decompose, resulting inwater and atomic oxygen. Like many other enzymes, HRP and some HRP-likeactivities can be inhibited by excess substrate. The complex formedbetween HRP and excess hydrogen peroxide is catalytically inactive andin the absence of an electron donor (e.g. chromogenic substance) isreversibly inhibited. It is the excess hydrogen peroxide and the absenceof an electron donor that brings about quenching of endogenous HRPactivities. When used in assay systems, HRP can also be used to converta defined substrate into its activated chromagen, thus causing a colorchange. The HRP enzyme can be conjugated to an antibody, peptide,polymer, or other molecule by a number of methods that are known in theart. Adding glutaraldehyde to a solution containing an admixture of HRPand antibody will result in more antibody molecules being conjugated toeach other than to the enzyme. In the two-step procedure, HRP reactswith the bifunctional reagents first. In the second stage, onlyactivated HRP is admixed with the antibody, resulting in much moreefficient labeling and no polymerization. HRP is also conjugated to(strept)avidin using the two-step glutaraldehyde procedure. This form isused in procedures where LAB and LSAB are substrates, for example.Conjugation with biotin also involves two steps, as biotin must first bederivatized to the biotinyl-N-hydroxysuccinimide ester or to biotinhydrazide before it can be reacted with the epsilonamino groups of theHRP enzyme.

3,3′-diaminobenzidine (DAB) is a substrate for enzymes such as HRP thatproduces a brown end product that is highly insoluble in alcohol andother organic solvents. Oxidation of DAB also causes polymerization,resulting in the ability to react with osmium tetroxide, and thusincreasing its staining intensity and electron density. Of the severalmetals and methods used to intensify the optical density of polymerizedDAB, gold chloride in combination with silver sulfide appears to be themost successful.

3-Amino-9-ethylcarbazole (AEC), is a substrate for enzymes such as HRP,and upon oxidation, forms a rose-red end product that is alcoholsoluble. Therefore, specimens processed with AEC must not be immersed inalcohol or alcoholic solutions (e.g., Harris' hematoxylin). Instead, anaqueous counterstain and mounting medium should be used.

4-Chloro-1-naphthol (CN) is a substrate for enzymes such as HRP thatprecipitates as a blue end product. Because CN is soluble in alcohol andother organic solvents, the specimen must not be dehydrated, exposed toalcoholic counterstains, or coverslipped with mounting media containingorganic solvents. Unlike DAB, CN tends to diffuse from the site ofprecipitation.

p-Phenylenediamine dihydrochloride/pyrocatechol (Hanker-Yates reagent)is a substrate for enzymes such as HRP that gives a blue-black reactionproduct that is insoluble in alcohol and other organic solvents. Likepolymerized DAB, this reaction product can be osmicated. Varying resultshave been achieved with Hanker-Yates reagent in immunoperoxidasetechniques.

Calf intestine alkaline phosphatase (AP) (molecular weight 100 kD)removes (by hydrolysis) and transfers phosphate groups from organicesters by breaking the P-0 bond; an intermediate enzyme-substrate bondis briefly formed. The chief metal activators for AP are Mg++, Mn++ andCa++.

AP had not been used extensively in immunohistochemistry untilpublication of the unlabeled alkaline phosphatase-anti-alkalinephosphatase (APAAP) procedure. The soluble immune complexes utilized inthis procedure have molecular weights of approximately 560 kD. The majoradvantage of the APAAP procedure compared to the peroxidaseanti-peroxidase (PAP) technique is the lack of interference posed byendogenous peroxidase activity. Endogenous peroxidase can be blockedusing a dilute solution of hydrogen peroxide. Because of the potentialdistraction of endogenous peroxidase activity on PAP staining, the APAAPtechnique is recommended for use on blood and bone marrow smears.Endogenous alkaline phosphatase activity from bone, kidney, liver andsome white cells can be inhibited by the addition of 1 mM levamisole tothe substrate solution, although 5 mM has been found to be moreeffective. Intestinal alkaline phosphatases are not adequately inhibitedby levamisole.

In the immunoalkaline phosphatase staining method, the enzyme hydrolyzesnaphthol phosphate esters (substrate) to phenolic compounds andphosphates. The phenols couple to colorless diazonium salts (chromogen)to produce insoluble, colored azo dyes. Several different combinationsof substrates and chromogens have been used successfully.

Naphthol AS-MX phosphate AP substrate can be used in its acid form or asthe sodium salt. The chromogen substrate Fast Red TR and Fast Blue BBproduce a bright red or blue end product, respectively. Both are solublein alcoholic and other organic solvents, so aqueous mounting media mustbe used. Fast Red TR is preferred when staining cell smears.

Additional exemplary substrates include naphthol AS-BI phosphate,naphthol AS-TR phosphate and 5-bromo-4-chloro-3-indoxyl phosphate(BCIP). Other possible chromogens include Fast Red LB, Fast Garnet GBC,Nitro Blue Tetrazolium (NBT) and iodonitrotetrazolium Violet (INT), forexample.

X. Immunodetection Methods

In still further embodiments, the present invention concernsimmunodetection methods for binding, purifying, removing, quantifyingand/or otherwise generally detecting biological components such as aligand as contemplated by the present invention. The antibodies preparedin accordance with the present invention may be employed. Someimmunodetection methods include immunohistochemistry, flow cytometry,enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA),immunoradiometric assay, fluoroimmunoassay, chemiluminescent assay,bioluminescent assay, and Western blot to mention a few. The steps ofvarious useful immunodetection methods have been described in thescientific literature, such as, e.g., Doolittle M H and Ben-Zeev O,Methods Mol Biol. 1999; 109:215-37; Gulbis B and Galand P. Hum Pathol.1993 December; 24(12):1271-85; and De Jager R et al., Semin Nucl Med.1993 April; 23(2): 165-79, each incorporated herein by reference.

In general, the immunobinding methods include obtaining a samplesuspected of comprising ligand protein, polypeptide and/or peptide, andcontacting the sample with a first ligand binding agent (e.g., ananti-ligand antibody) in accordance with the present invention, as thecase may be, under conditions effective to allow the formation ofimmunocomplexes.

In terms of antigen detection, the biological sample analyzed may be anysample in which it is desirable to detect FOLR1 such as fluidic extract,blood, plasma, serum, spinal fluid, lymph fluid, tissue section orspecimen, homogenized tissue extract, biopsy aspirates, a cell,separated and/or purified forms FOLR1-containing compositions, or anybiological fluid. In some embodiments, blood, plasma, or lymph samplesor extracts are used.

Contacting the chosen biological sample with the antibody undereffective conditions and for a period of time sufficient to allow theformation of immune complexes (primary immune complexes) is generally amatter of simply adding the antibody composition to the sample andincubating the mixture for a period of time long enough for theantibodies to form immune complexes with, i.e., to bind to, any ligandprotein antigens present. After this time, the sample-antibodycomposition, such as a tissue section, ELISA plate, dot blot or westernblot, will generally be washed to remove any non-specifically boundantibody species, allowing only those antibodies specifically boundwithin the primary immune complexes to be detected.

In general, the detection of immunocomplex formation is well known inthe art and may be achieved through the application of numerousapproaches. These methods are generally based upon the detection of alabel or marker, such as any of those radioactive, fluorescent,biological and enzymatic tags. U.S. patents concerning the use of suchlabels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350;3,996,345; 4,277,437; 4,275,149 and 4,366,241, each incorporated hereinby reference. Of course, one may find additional advantages through theuse of a secondary binding ligand such as a second antibody and/or abiotin/avidin ligand binding arrangement, as is known in the art.

The anti-ligand antibody employed in the detection may itself be linkedto a detectable label, wherein one would then simply detect this label,thereby allowing the amount of the primary immune complexes in thecomposition to be determined. Alternatively, the first antibody thatbecomes bound within the primary immune complexes may be detected bymeans of a second binding agent that has binding affinity for theantibody. In these cases, the second binding agent may be linked to adetectable label. The second binding agent is itself often an antibody,which may thus be termed a “secondary” antibody. The primary immunecomplexes are contacted with the labeled, secondary binding agent, orantibody, under effective conditions and for a period of time sufficientto allow the formation of secondary immune complexes. The secondaryimmune complexes are then generally washed to remove anynon-specifically bound labeled secondary antibodies or ligands, and theremaining label in the secondary immune complexes is then detected.

Further methods include the detection of primary immune complexes by atwo-step approach. A second binding agent, such as an antibody, that hasbinding affinity for the antibody is used to form secondary immunecomplexes, as described herein. After washing, the secondary immunecomplexes are contacted with a third binding agent or antibody that hasbinding affinity for the second antibody, again under effectiveconditions and for a period of time sufficient to allow the formation ofimmune complexes (tertiary immune complexes). The third ligand orantibody is linked to a detectable label, allowing detection of thetertiary immune complexes thus formed. This system may provide forsignal amplification if this is desired.

In another embodiment, a biotinylated monoclonal or polyclonal antibodyis used to detect the target antigen(s), and a second step antibody isthen used to detect the biotin attached to the complexed biotin. In thatmethod the sample to be tested is first incubated in a solutioncomprising the first step antibody. If the target antigen is present,some of the antibody binds to the antigen to form a biotinylatedantibody/antigen complex. The antibody/antigen complex is then amplifiedby incubation in successive solutions of streptavidin (or avidin),biotinylated DNA, and/or complementary biotinylated DNA, with each stepadding additional biotin sites to the antibody/antigen complex. Theamplification steps are repeated until a suitable level of amplificationis achieved, at which point the sample is incubated in a solutioncomprising the second step antibody against biotin. This second stepantibody is labeled, as for example with an enzyme that can be used todetect the presence of the antibody/antigen complex by histoenzymologyusing a chromogen substrate. With suitable amplification, a conjugatecan be produced that is macroscopically visible.

In one embodiment, immunohistochemistry (IHC) is used for immunologicaldetection. Using IHC, detection of FOLR1 in a sample can be achieved bytargeting a sample with a probe e.g., an anti-FOLR1 antibody. The probecan be linked, either directly or indirectly to a detectable label orcan be detected by another probe that is linked, either directly orindirectly to a detectable label.

In some embodiments, IHC can distinguish between different levels ofprotein expression, e.g., calibrated IHC. In some embodiments, the IHCcan distinguish staining intensity for samples having low FOLR1,intermediate FOLR1, or high FOLR1 expression.

In one embodiment, immunological detection (by immunohistochemistry) ofFOLR1 is scored for both intensity and uniformity (percent of stainedcells—membrane only). Comparative scales for FOLR1 expression forintensity correlate as 0—Negative, 0—1—Very Weak, 1—Weak, 1—2—Weak toModerate, 2—Moderate, 2—3—Moderate to Strong, 3—Strong, 3+—Very Strong.Quantitatively. Score 0 represents that no membrane staining isobserved. Score 1 represents that a faint/barely perceptible membranestaining is detected. For Score 2, a weak to moderate complete membranestaining is observed. Lastly, Score 3 (or 3+) represents that moderateto complete membrane staining is observed. Those samples with 0 or 1score for FOLR1 expression can be characterized as not having elevatedFOLR1 expression, whereas those samples with 2 or 3 scores can becharacterized as overexpressing or having elevated FOLR1. In anotherembodiment, using the antibodies, antigen-binding fragments thereof, orpolypeptides provided herein, those samples with a 0 score for FOLR1expression can be characterized as not having elevated FOLR1 expression,those samples with a 1 score can be characterized as having increasedexpression of FOLR1, and those samples with 2 or 3 scores can becharacterized as overexpressing or having elevated FOLR1.

Samples overexpressing FOLR1 can also be rated by immunohistochemicalscores corresponding to the number of copies of FOLR1 moleculesexpressed per cell, or antibodies bound per cell (ABC), and can beendetermined biochemically. Comparative scales for FOLR1 uniformity(percent cell membrane staining) are as follows: Negative=0; Focal=<25%;heterogeneous (hetero)=25-75%, and homogeneous (homo)=>75%.

In one embodiment, immunological detection (by immunohistochemistry) ofFOLR1 is scored using H-scores. H-scores combine staining intensityscores (e.g., a score of 0 to 3, wherein 0 represents no staining, and 3represents strong staining) with the percentage of cells that arepositive for membrane staining (i.e., uniformity). An H-score can becalculated as follows: H score=[0*(percentage of cells staining atintensity 0)]+[1*(percentage of cells staining at intensity1)]+[2*(percentage of cells staining at intensity 2)]+[3*(percentage ofcells staining at intensity 3)]. Accordingly, an H-score can range from0 (no cell membranes staining) to 300 (all cell membranse staining atintensity 3).

In one embodiment, a subject having cancer is identified as a candidatefor treatment with an anti-FOLR1 treatment regimen (e.g., IMGN853) whenthe H-score for FOLR expression in a tumor sample from the subject is atleast 50. In one embodiment, a subject having cancer is identified as acandidate for treatment with an anti-FOLR1 treatment regimen (e.g.,IMGN853) when the H-score for FOLR expression in a tumor sample from thesubject is at least 75. In one embodiment, a subject having cancer isidentified as a candidate for treatment with an anti-FOLR1 treatmentregimen (e.g., IMGN853) when the H-score for FOLR expression in a tumorsample from the subject is at least 100. In one embodiment, a subjecthaving cancer is identified as a candidate for treatment with ananti-FOLR1 treatment regimen (e.g., IMGN853) when the H-score for FOLRexpression in a tumor sample from the subject is at least 125. In oneembodiment, a subject having cancer is identified as a candidate fortreatment with an anti-FOLR1 treatment regimen (e.g., IMGN853) when theH-score for FOLR expression in a tumor sample from the subject is atleast 150. In one embodiment, a subject having cancer is identified as acandidate for treatment with an anti-FOLR1 treatment regimen (e.g.,IMGN853) when the H-score for FOLR expression in a tumor sample from thesubject is at least 175. In one embodiment, a subject having cancer isidentified as a candidate for treatment with an anti-FOLR1 treatmentregimen (e.g., IMGN853) when the H-score for FOLR expression in a tumorsample from the subject is at least 200. In another embodiment, asubject having cancer is identified as a candidate for treatment with ananti-FOLR1 regimen (e.g., IMGN853) when the H-score for FOLR expressionin a tumor sample from the subject is at least 225. In anotherembodiment, a subject having cancer is identified as a candidate fortreatment with an anti-FOLR1 regimen (e.g., IMGN853) when the H-scorefor FOLR expression in a tumor sample from the subject is at least 250.In another embodiment, a subject having cancer is identified as acandidate for treatment with an anti-FOLR1 regimen (e.g., IMGN853) whenthe H-score for FOLR expression in a tumor sample from the subject is atleast 275. In another embodiment, a subject having cancer is identifiedas a candidate for treatment with an anti-FOLR1 regimen (e.g., IMGN853)when the H-score for FOLR expression in a tumor sample from the subjectis 300.

In another embodiment, a subject having ovarian cancer is identified asa candidate for treatment with an anti-FOLR1 regimen (e.g., IMGN853)when the H-score for FOLR expression in an ovarian tumor sample from thesubject is 75 to 300. In another embodiment, a subject having ovariancancer is identified as a candidate for treatment with an anti-FOLR1regimen (e.g., IMGN853) when the H-score for FOLR expression in anovarian tumor sample from the subject is at least 75. In anotherembodiment, a subject having ovarian cancer is identified as a candidatefor treatment with an anti-FOLR1 regimen (e.g., IMGN853) when theH-score for FOLR expression in an ovarian tumor sample from the subjectis at least 100. In another embodiment, a subject having ovarian canceris identified as a candidate for treatment with an anti-FOLR1 regimen(e.g., IMGN853) when the H-score for FOLR expression in an ovarian tumorsample from the subject is at least 125. In another embodiment, asubject having ovarian cancer is identified as a candidate for treatmentwith an anti-FOLR1 regimen (e.g., IMGN853) when the H-score for FOLRexpression in an ovarian tumor sample from the subject is at least 150.In another embodiment, a subject having ovarian cancer is identified asa candidate for treatment with an anti-FOLR1 regimen (e.g., IMGN853)when the H-score for FOLR expression in an ovarian tumor sample from thesubject is at least 175. In another embodiment, a subject having ovariancancer is identified as a candidate for treatment with an anti-FOLR1regimen (e.g., IMGN853) when the H-score for FOLR expression in anovarian tumor sample from the subject is at least 200. In anotherembodiment, a subject having ovarian cancer is identified as a candidatefor treatment with an anti-FOLR1 regimen (e.g., IMGN853) when theH-score for FOLR expression in an ovarian tumor sample from the subjectis at least 225. In another embodiment, a subject having ovarian canceris identified as a candidate for treatment with an anti-FOLR1 regimen(e.g., IMGN853) when the H-score for FOLR expression in an ovarian tumorsample from the subject is at least 250. In another embodiment, asubject having ovarian cancer is identified as a candidate for treatmentwith an anti-FOLR1 regimen (e.g., IMGN853) when the H-score for FOLRexpression in an ovarian tumor sample from the subject is at least 275.In another embodiment, a subject having ovarian cancer is identified asa candidate for treatment with an anti-FOLR1 regimen (e.g., IMGN853)when the H-score for FOLR expression in an ovarian tumor sample from thesubject is 300.

In another embodiment, a subject having NSCLC is identified as acandidate for treatment with an anti-FOLR1 regimen (e.g., IMGN853) whenthe H-score for FOLR expression in an NSCLC tumor sample from thesubject is 50 to 300. In another embodiment, a subject having NSCLC isidentified as a candidate for treatment with an anti-FOLR1 regimen(e.g., IMGN853) when the H-score for FOLR expression in an NSCLC tumorsample from the subject is at least 50. In another embodiment, a subjecthaving NSCLC is identified as a candidate for treatment with ananti-FOLR1 regimen (e.g., IMGN853) when the H-score for FOLR expressionin an NSCLC tumor sample from the subject is at least 75. In anotherembodiment, a subject having NSCLC is identified as a candidate fortreatment with an anti-FOLR1 regimen (e.g., IMGN853) when the H-scorefor FOLR expression in an NSCLC tumor sample from the subject is atleast 100. In another embodiment, a subject having NSCLC is identifiedas a candidate for treatment with an anti-FOLR1 regimen (e.g., IMGN853)when the H-score for FOLR expression in an NSCLC tumor sample from thesubject is at least 125. In another embodiment, a subject having NSCLCis identified as a candidate for treatment with an anti-FOLR1 regimen(e.g., IMGN853) when the H-score for FOLR expression in an NSCLC tumorsample from the subject is at least 150. In another embodiment, asubject having NSCLC is identified as a candidate for treatment with ananti-FOLR1 regimen (e.g., IMGN853) when the H-score for FOLR expressionin an NSCLC tumor sample from the subject is at least 175. In anotherembodiment, a subject having NSCLC is identified as a candidate fortreatment with an anti-FOLR1 regimen (e.g., IMGN853) when the H-scorefor FOLR expression in an NSCLC tumor sample from the subject is atleast 200. In another embodiment, a subject having NSCLC is identifiedas a candidate for treatment with an anti-FOLR1 regimen (e.g., IMGN853)when the H-score for FOLR expression in an NSCLC tumor sample from thesubject is at least 225. In another embodiment, a subject having NSCLCis identified as a candidate for treatment with an anti-FOLR1 regimen(e.g., IMGN853) when the H-score for FOLR expression in an NSCLC tumorsample from the subject is at least 250. In another embodiment, asubject having NSCLC is identified as a candidate for treatment with ananti-FOLR1 regimen (e.g., IMGN853) when the H-score for FOLR expressionin an NSCLC tumor sample from the subject is at least 275. In anotherembodiment, a subject having NSCLC is identified as a candidate fortreatment with an anti-FOLR1 regimen (e.g., IMGN853) when the H-scorefor FOLR expression in an NSCLC tumor sample from the subject is 300.

In another embodiment, a subject having endometrial cancer is identifiedas a candidate for treatment with an anti-FOLR1 regimen (e.g., IMGN853)when the H-score for FOLR expression in an endometrial tumor sample fromthe subject is 50 to 300. In another embodiment, a subject havingendometrial cancer is identified as a candidate for treatment with ananti-FOLR1 regimen (e.g., IMGN853) when the H-score for FOLR expressionin an endometrial tumor sample from the subject is at least 50. Inanother embodiment, a subject having endometrial cancer is identified asa candidate for treatment with an anti-FOLR1 regimen (e.g., IMGN853)when the H-score for FOLR expression in an endometrial tumor sample fromthe subject is at least 75. In another embodiment, a subject havingendometrial cancer is identified as a candidate for treatment with ananti-FOLR1 regimen (e.g., IMGN853) when the H-score for FOLR expressionin an endometrial tumor sample from the subject is at least 100. Inanother embodiment, a subject having endometrial cancer is identified asa candidate for treatment with an anti-FOLR1 regimen (e.g., IMGN853)when the H-score for FOLR expression in an endometrial tumor sample fromthe subject is at least 125. In another embodiment, a subject havingendometrial cancer is identified as a candidate for treatment with ananti-FOLR1 regimen (e.g., IMGN853) when the H-score for FOLR expressionin an endometrial tumor sample from the subject is at least 150. Inanother embodiment, a subject having endometrial cancer is identified asa candidate for treatment with an anti-FOLR1 regimen (e.g., IMGN853)when the H-score for FOLR expression in an endometrial tumor sample fromthe subject is at least 175. In another embodiment, a subject havingendometrial cancer is identified as a candidate for treatment with ananti-FOLR1 regimen (e.g., IMGN853) when the H-score for FOLR expressionin an endometrial tumor sample from the subject is at least 200. Inanother embodiment, a subject having endometrial cancer is identified asa candidate for treatment with an anti-FOLR1 regimen (e.g., IMGN853)when the H-score for FOLR expression in an endometrial tumor sample fromthe subject is at least 225. In another embodiment, a subject havingendometrial cancer is identified as a candidate for treatment with ananti-FOLR1 regimen (e.g., IMGN853) when the H-score for FOLR expressionin an endometrial tumor sample from the subject is at least 250. Inanother embodiment, a subject having endometrial cancer is identified asa candidate for treatment with an anti-FOLR1 regimen (e.g., IMGN853)when the H-score for FOLR expression in an endometrial tumor sample fromthe subject is at least 275. In another embodiment, a subject havingendometrial cancer is identified as a candidate for treatment with ananti-FOLR1 regimen (e.g., IMGN853) when the H-score for FOLR expressionin an endometrial tumor sample from the subject is 300.

By way of example, an H-score in a subject having ovarian cancer may beas follows:

H score=(75% at intensity 0)+(0% at intensity 1)+(0% at intensity2)+(25% at intensity 3)=75; or

H score=(0% at intensity 0)+(75% at intensity 1)+(0% at intensity2)+(25% at intensity 3)=150.

In another example, an H-score in a subject having endometrial cancermay be as follows:

H score=(75% at intensity 0)+(0% at intensity 1)+(25% at intensity2)+(0% at intensity 3)=50; or

H score=(0% at intensity 0)+(75% at intensity 1)+(25% at intensity2)+(0% at intensity 3)=125.

In all four examples above, the subject could be identified as acandidate for treatment with an anti-FOLR1 treatment regimen (e.g.,IMGN853).

In one embodiment, immunological detection (by immunohistochemistry) ofFOLR1 is scored using percent positivity and intensity across a sample.In this embodiment, selection for treatment with an anti-FOLR1 treatmentregimen is based on the percentage of cells in a sample that are foundto express membrane FOLR1 at a specified level that reflects both thestaining intensity (e.g., 1, 2, or 3) and uniformity (e.g.,heterogeneous or homogeneous (see Table 11)). For example, a samplehaving at least 25% (i.e., 25-75% or >75%) of the cells staining forFOLR1 positivity at 3 could be characterized as “3 hetero” and “3 homo”or, collectively, as “at least 25% positive at 3.”

In one embodiment, a subject having ovarian cancer is identified as acandidate for treatment with an anti-FOLR1 treatment regimen (e.g.,IMGN853) when at least 25% of the FOLR1 membrane expression in a tumorsample from the subject has an intensity score of 3 by IHC. In oneembodiment, the IHC is performed using the FOLR1-2.1 antibody.

In another embodiment, a subject having endometrial cancer is identifiedas a candidate for treatment with an anti-FOLR1 treatment regimen (e.g.,IMGN853) when at least 25% of the FOLR membrane expression in a tumorsample from the subject has an intensity score of at least 2 by IHC. Inone embodiment, the IHC is performed using the FOLR1-2.1 antibody.

In another embodiment, a subject having NSCLC is identified as acandidate for treatment with an anti-FOLR1 treatment regimen (e.g.,IMGN853) when at least 25% of the FOLR membrane expression in a tumorsample from the subject has an intensity score of at least 2 by IHC. Inone example, the IHC is peformed using the FOLR1-2.1 antibody for IHC.

IHC can be performed manually or using an automated system (e.g., usingan automated stainer). IHC can be performed on cells, cell pellets,tissues, preparations from blood, plasma, serum, or lymph fluid, etc. Insome embodiments, the samples are fixed samples. In some embodiments,the samples are paraffin embedded samples. In some embodiments, thesamples are formalin fixed and paraffin embedded samples.

In one embodiment, flow cytometry is used for immunological detection.Thus, for example, the number of antibodies bound per cell (ABC) can beassessed using flow cytometry. A high number of anti-FOLR1 antibodiesbound per cell can indicate high FOLR1 expression levels and a highlikelihood to be susceptible to treatment with an anti-FOLR1 antibody orimmunoconjugate thereof.

XI. Compositions and Kits

Also provided by the invention are compositions and kits for use in thepractice of the present invention as disclosed herein. Such kits maycomprise containers, each with one or more of the various reagents(typically in concentrated form) utilized in the methods, including, forexample, one or more binding agents (antibodies), already attached to amarker or optionally with reagents for coupling a binding agent to anantibody (as well as the marker itself), buffers, and/or reagents andinstrumentation for the isolation (optionally by microdissection) tosupport the practice of the invention. A label or indicator describing,or a set of instructions for use of, kit components in a liganddetection method of the present invention, will also be typicallyincluded, where the instructions may be associated with a package insertand/or the packaging of the kit or the components thereof.

In still further embodiments, the present invention concernsimmunodetection kits for use with the immunodetection methods describedherein. As the antibodies are generally used to detect FOLR1, heantibodies will generally be included in the kit. The immunodetectionkits will thus comprise, in suitable container means, a first antibodythat binds to FOLR1 and/or optionally, an immunodetection reagent and/orfurther optionally, a FOLR1 protein or cell sample containing FOLR1.

The immunodetection reagents of the kit may take any one of a variety offorms, including those detectable labels that are associated with and/orlinked to the given antibody. Detectable labels that are associated withand/or attached to a secondary binding ligand are also contemplated.Exemplary secondary ligands are those secondary antibodies that havebinding affinity for the first antibody.

Further suitable immunodetection reagents for use in the present kitsinclude the two-component reagent that comprises a secondary antibodythat has binding affinity for the first antibody, along with a thirdantibody that has binding affinity for the second antibody, the thirdantibody being linked to a detectable label. As noted herein, a numberof exemplary labels are known in the art and/or all such labels may besuitably employed in connection with the present invention.

The kits may further comprise a therapeutic agent for the treatment ofcancer, such as an anti-FOLR1 immunoconjugate.

The kit may further comprise an a FOLR1 detection reagent used tomeasure FOLR1 expression in a subject comprising a FOLR1 detectionreagent, and instructions for use. In one embodiment, the FOLR1detection reagent comprises a FOLR1 binding peptide or anti-FOLR1antibody. In another embodiment, the kit further comprises a secondaryantibody which binds the anti-FOLR1 antibody.

In one embodiment the FOLR1-specific antibody is included at aconcentration of about 0.1 to about 20 μg/mL, about 0.1 to about 15μg/mL, about 0.1 to about 10 μg/mL, about 0.5 to about 20 μg/mL, about0.5 to about 15 μg/mL, about 0.5 to about 10 μg/mL, about 1 to about 20μg/mL, about 1 to about 15 μg/mL, about 1 to about 10 μg/mL, about 2 toabout 20 μg/mL, about 2 to about 15 μg/mL, or about 2 to about 10 μg/mL.In another embodiment, the FOLR1-specific antibody is included at aconcentration of about 1.5 μg/mL, about 2 μg/mL, about 3 μg/mL, about 4μg/mL, about 5 μg/mL, about 6 μg/mL, about 7 μg/mL, about 8 μg/mL, about9 μg/mL, or about 10 μg/mL. In another embodiment, the FOLR1-specificantibody is included at a concentration of about 2 μg/mL. In anotherembodiment, the FOLR1-specific antibody is included at a concentrationof about 10 μg/mL.

In another embodiment, the antibody is included in concentrated solutionwith instructions for dilutions to achieve a final concentration ofabout 1 to about 20 μg/mL, about 1 to about 15 μg/mL, about 1 to about10 μg/mL, about 2 to about 20 μg/mL, about 2 to about 15 μg/mL, or about2 to about 10 μg/mL. In another embodiment, the antibody is included inconcentrated solution with instructions for dilutions to achieve a finalconcentration of about 1.5 μg/mL, about 2 μg/mL, about 3 μg/mL, about 4μg/mL, about 5 μg/mL, about 6 μg/mL, about 7 μg/mL, about 8 μg/mL, about9 μg/mL, or about 10 μg/mL. In another embodiment, the antibody isincluded in concentrated solution with instructions for dilutions toachieve a final concentration of about 2 μg/mL. In another embodiment,the antibody is included in concentrated solution with instructions fordilutions to achieve a final concentration of about 10 μg/ml.

In another embodiment, the kit further comprises a detection reagentselected from the group consisting of: an enzyme, a fluorophore, aradioactive label, and a luminophore. In another embodiment, thedetection reagent is selected from the group consisting of: biotin,digoxigenin, fluorescein, tritium, and rhodamine.

The kit can also include instructions for detection and scoring of FOLR1expression. The kit can also include control or reference samples.Non-limiting examples of control or reference samples include cellpellets or tissue culture cell lines derived from normal (normalcontrol) or tumor (positive control) samples. Exemplary cell linesinclude cell lines stably or transiently transfected with an expressionvector that expresses FOLR1. Additional examples include cell pelletsand tissue samples described in the Examples.

In some embodiments, a kit is a packaged combination including the basicelements of: (a) capture reagents comprised of the monoclonal antibodiesagainst human FOLR1; and (b) detection reagents which can also compriseFOLR1 monoclonal antibodies, but can also comprise detectable (labeledor unlabeled) antibodies that bind to FOLR1. These basic elements aredefined herein.

In one embodiment, the kit further comprises a solid support for thecapture reagents, which can be provided as a separate element or onwhich the capture reagents are already immobilized. Hence, the captureantibodies in the kit can be immobilized on a solid support, or they canbe immobilized on such support that is included with the kit or providedseparately from the kit.

In one embodiment, the capture reagent is coated on a microtiter plate.The detection reagent can be labeled antibodies detected directly orunlabeled antibodies that are detected by labeled antibodies directedagainst the unlabeled antibodies raised in a different species. Wherethe label is an enzyme, the kit will ordinarily include substrates andcofactors required by the enzyme, and where the label is a fluorophore,a dye precursor that provides the detectable chromophore. Where thedetection reagent is unlabeled, the kit can further comprise a detectionmeans for the detectable antibodies, such as the labeled antibodiesdirected to the unlabeled antibodies, e.g., in a fluorimetric-detectedformat. Where the label is an enzyme, the kit will ordinarily includesubstrates and cofactors required by the enzyme, where the label is afluorophore, a dye precursor that provides the detectable chromophore,and where the label is biotin, an avidin such as avidin, streptavidin,or streptavidin conjugated to HRP or β-galactosidase with MUG.

In one embodiment, the capture reagent is the FOLR1 antibody 2.1, 5.7,or 9.20 or an antibody comprising the sequences of antibody 2.1, 5.7 or9.20. In one embodiment, the detection reagent is the FOLR1 antibody2.1, 5.7, or 9.20 or an antibody comprising the sequences of antibody2.1, 5.7 or 9.20. In another embodiment, the detection reagent FOLR1antibody 2.1, 5.7, or 9.20 or an antibody comprising the sequences ofantibody 2.1, 5.7 or 9.20 is biotinylated.

The kit also typically contains instructions for carrying out the assay,and/or FOLR1 protein, or fragments thereof (e.g., FOLR1 extracellulardomain or the FOLR1 extracellular domain and all or a part of the GPIlinkage domain) as an antigen standard, as well as other additives suchas stabilizers, washing and incubation buffers, and the like. In oneembodiment, the FOLR1 antigen standard is a FOLR1-Fc immunoadhesin. Thekit can also include instructions for detection and scoring of FOLR1expression.

The components of the kit can be provided in predetermined ratios, withthe relative amounts of the various reagents suitably varied to providefor concentrations in solution of the reagents that substantiallymaximize the sensitivity of the assay. Particularly, the reagents can beprovided as dry powders, usually lyophilized, including excipients,which on dissolution will provide for a reagent solution having theappropriate concentration for combining with the sample to be tested.

Compositions comprising the antibodies or antigen-binding fragmentsdescribed herein are also provided. In one embodiment, a compositioncomprises an anti-FOLR1 antibody or antigen-binding fragment describedherein and a buffer, e.g., a buffer that can be used in a detectionassay such as FACS, IHC, or ELISA. Such buffers are known to those ofordinary skill in the art and include diluents. By way of example,certain FACS buffers are provided herein, e.g., in the working examples.FACS buffers can also contain, for example, serum or albumin (such ascalf serum, goat serum, or BSA) and/or sodium azide. FACS buffers canalso contain PBS, EDTA, and/or DNAse or any combination thereof. IHCbuffers are also provided herein and known to those of ordinary skill inthe art. IHC buffers can contain, for example, casein serum or albumin(such as calf serum, goat serum, or BSA), Tween or Triton, PBS and/orsodium azide or any combination thereof. ELISA buffers are also providedherein and known to those of ordinary skill in the art. ELISA bufferscan contain, for example, serum or albumin (such as calf serum, goatserum, or BSA), non-fat dry milk, casein, and/or gelatin or anycombination thereof.

Embodiments of the present disclosure can be further defined byreference to the following non-limiting examples, which describe indetail preparation of certain antibodies of the present disclosure andmethods for using antibodies of the present disclosure. It will beapparent to those skilled in the art that many modifications, both tomaterials and methods, can be practiced without departing from the scopeof the present disclosure.

EXAMPLES

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application.

Example 1: Generation of FOLR1 Hybridomas

Hybridomas producing anti-human FOLR1 monoclonal antibodies that aresuitable for immunohistochemistry (IHC) staining (antibodies of theinvention) were selected from more than 16,000 hybridomas. Thehybridomas were produced by immunization of wild-type Balb/c mice withdifferent antigens including: formalin fixed 300-19 cells that have beentransfected with human FOLR1, human FOLR1-murine IgG2a Fc recombinantprotein, and human FOLR1 recombinant protein. The immunization withfixed 300-19 cells was conducted by subcutaneous injection oftransfected 300-19 cells in PBS (5E6 cells/mouse/injection) in theabsence of any adjuvant. The immunization with FOLR1 recombinantproteins was done by subcutaneous injection of the protein emulsified incomplete Freund's adjuvant (CFA) or incomplete Freund's adjuvant forboost (Sigma) or Magic mouse adjuvant (Creative Diagnostics). Generally,mice were immunized five times with two week intervals before receivinga final boost by intraperitoneal injection of the immunogen three daysprior to fusion.

A total of 16 independent fusions (including fusions 352, 353, and 354)were carried out using spleen cells that originated from the immunizedwild-type Balb/c mice and murine myeloma P3×63Ag8.653 cells (P3 cells).Cell fusion was conducted using an ECM200 electrofusion machine (BTXHarvard Apparatus) according to standard protocols. Each fusion yieldedmore than 1,000 hybridomas. Antibodies produced by these hybridomas werescreened and confirmed by a FACS based method using denaturedFOLR1-positive and FOLR1-negative cells. Of the greater than 16,000hybridomas screened, 14 hybridomas that were positive by FACS screeningwere discovered. All of the positive hybridomas originated from miceimmunized with human FOLR1-murine IgG2a Fc recombinant protein.

Of the 14 hybridomas which were initially positive by FACS screening,only ten showed a sufficient IgG concentration for further analysis.

Example 2. Immunohistochemical Evaluation of Hybridoma Supernatants

Ten of the initial 14 hybridomas were analyzed by IHC. The analysis wasperformed using the Leica Bond RX Automated Stainer and the reagents andconditions listed in Table 9.

TABLE 9 IHC Reagents and Assay Conditions Step Action/Reagent (Vendor)Tune Bake Temperature: 60° C. 30 Minutes Dewax Bond Dewax Solution(Leica) Fixed 100% Ethanol (Pharmco Aaper) Antigen Retrieval BondEpitope Retrieval 2 20 Minutes (ethylenediaminetetraacetic acid based pH9.0 solution) Endogenous Peroxidase Peroxide (Leica)  5 Minutes BlockTest Article ImmunoGen, Inc. generated 15 Minutes antibodies at varyingconcentrations prepared by diluting in Leica Antibody Diluent DetectionPost Primary Regent (Leica)  8 Minutes Polymer (Leica)  8 Minutes MixedDAB (Leica) 10 Minutes Counterstain Hematoxylin (Leica)  5 Minutes

Slides containing formalin fixed paraffin embeded (FFPE) cells, normaltissues, patient lung tumor biopsies, and patient ovarian tumor biopsieswere baked at 60° C. and dewaxed using Bond Dewax Solution and 100%Ethanol. Heat induced epitope retrieval using Bond Epitope Retrieval 2(ethylenediaminetetraacetic acid based pH 9.0 solution) was performedfor 20 minutes and endogenous peroxidase was blocked with peroxide for 5minutes. Slides were incubated with ImmunoGen, Inc. generated antibodiesor Leica/Novocastra muIgG1 control antibodies at varying concentrationsfor 15 minutes. Bound antibodies were detected by incubation with theLeica Bond Refine detection system. Following the application of theantibodies, slides were incubated with Post Primary Reagent (rabbitanti-mouse IgG) for 8 minutes, Polymer (goat anti-rabbit polymer) for 8minutes, and DAB (3,3-diaminobenzidine tetrahydrochloride) for 10minutes which resulted in a brown color signal. Slides werecounterstained with hematoxylin for 5 minutes.

FFPE tissue samples were derived from human tissue blocks obtained fromProteogenex and the Cooperative Human Tissue Network (CHTN) as outlinedbelow. FFPE cell samples were derived from the KB cell line supplied byAmerican Tissue Culture Collection. Slides containing sections ofsamples were prepared from FFPE blocks using a microtome set at 5 μm andwere mounted on positively charged slides. These slides were allowed toair dry overnight prior to staining.

TABLE 10 FFPE Test Samples Human Tissue Type Commercial Source NormalLung CHTN Normal Pancreas CHTN Normal Salivary Gland CHTN OvarianPapillary Serous Adenocarcinoma Proteogenex Lung Adenocarcinoma CHTN

FOLR1 staining intensity and distribution patterns were scored relativeto control IgG staining (non-specific). Intensity was scored on a scaleof 0 to 3 where 0=no staining, 1=weak staining, 2=moderate staining, and3=strong staining. Uniformity of the staining was scored as negative (nocells exhibit positive staining), focal (<25% of cells stained),heterogeneous (25-75% of cells stained), and homogeneous (>75% of cellsstained). The staining intensity and scoring scales are described below.All staining was evaluated by a Board certified pathologist.

TABLE 11 Intensity and Uniformity of Staining Intensity (Amount ofUniformity (Percent Membrane Staining) of Positive Cells) 0 Negative 0Negative 1 Weak Focal <25% 2 Moderate Heterogeneous (hetero) 25-75% 3Strong Homogeneous (homo) >75%

IHC Selection Process to Identify Hybridoma(s) for FFPE FOLR1 IHC

Primary clones positive by FACS on FOLR1-positive denatured cells (tenclones total) were evaluated by IHC. Two clones were obtained fromfusion 352 (clones 352.1 and 352.2). Six clones were obtained fromfusion 353 (clones 353.1, 353.2, 353.3, 353.5, 353.9, 353.15), and twoclones were obtained from fusion 354 (clones 354.1 and 354.2). Hybridomasupernatants were collected from the cultured hybridoma cells and usedfor the analysis. Antibody concentrations in hybridoma supernatants weredetermined by ELISA using anti-murine L-chain specific polyclonalantibody to capture murine monoclonal antibody from supernatant andanti-murine Fc-specific polyclonal antibody to detect the capturedantibody; murine monoclonal IgG1 sample with know concentration was usedas a standard to calculate IgG concentration. Cell culture media(undiluted) was shown not to interfere with IHC staining methods (nobackground/non-specific staining was noted when media was used in placeof the primary antibody). Ten supernatants (IMGN 352.1, 352.2, 353.1,353.2, 353.3, 353.5, 353.9, 353.15, 354.1, and 354.2) diluted at varyingconcentrations up to 10 μg/mL in Leica Antibody Diluent were stainedusing FOLR1 known positive control samples (human normal lung, patientderived ovarian serous papillary adenocarcinoma, and KB cells) andevaluated to identify positive candidate clones (clones depictingacceptable membrane staining and specificity in FOLR1 positive samples).Five of the ten clones exhibited acceptable membrane staining in FOLR1positive samples and good specificity. A suitable staining concentrationwas experimentally determined for each of the five candidate clones asfollows: 353.1 (0.7 μg/mL), 353.2 (2.3 μg/mL), 353.3 (2.3 μg/mL), 353.5(2 μg/mL and 10 μg/mL), and 353.9 (2 μg/mL and 10 μg/mL). Of theremaining 5 clones, clone 353.15 (stained at 2 and 10 μg/mL) exhibitedacceptable membrane staining in KB cells and normal lung tissue;however, cytoplasmic staining only was observed in the patient ovariantumor tissue tested. Clones 352.1, 352.2, and 354.1 exhibited no visiblestaining in any samples and clone 354.2 exhibited only apparentnon-specific cytoplasmic staining, all considered unacceptable.

The five candidate clones were further subcloned. Subclones for fourclones (353.2, 353.3, 353.5 and 353.9) were successfully identified nosubclones of clone 353.1 was generated. A total of eight subclones werepurified. Two subclones were obtained from clone 353.5 (353.5-7 and353.5-10). Two subclones were obtained from clone 353.9 (353.9-20 and353.9-21). Two subclones were obtained from clone 353.3 (353.3-8 and353.3-9), and two subclones were obtained from clone 353.2 (353.2-1 and353.2-12). (Note that these subclones are also referred to as 5.7, 5.10,9.20, 9.21, 3.8, 3.9, 2.1, and 2.12, respectively.) IHC characterizationof the subclones was performed using methods described above (Table 9:IHC Reagents and Assay Conditions) at antibody concentrations of 2 and10 μg/mL. The eight subclones were also sequenced as described inExample 3, below. The candidate subclones were further evaluated toidentify and rank for optimal membrane staining and specificity asfollows: [353.2-1, 353.2-12], [353.9-20, 353.9-21], [353.5-7,353.5-10],and [353.3-8, 353.3-9] and were selected for further characterization(subclones are bracketed together according to sequence identity asdescribed in Example 3).

Two subclones were selected for further IHC assay optimization: 353.2-1and 353.9-20. Both antibodies were used to stain human normal lung,human normal salivary gland, human normal pancreas, patient ovariancancer biopsies, patient non-small cell lung cancer (NSCLC) biopsies,and patient clear cell renal cell carcinoma biopsies. At optimalconditions (see Table 12 below and FIG. 13), both subclones exhibitedspecific and appropriately sensitive staining in both human normal andpatient tumor tissues. Ducts of pancreas, respiratory epithelium ofnormal lung, and intercalated ducts exhibited positive membraneassociated staining. Acinar cells/islets of pancreas, interalveolarconnective tissue of lung, and acinar cells of salivary gland expectedto be negative did not exhibit positive staining with either subclone.Tumor cells from ovarian cancer, NSCLC, and clear cell renal cellcarcinoma samples expected to be positive exhibited positive membraneassociated staining that was localized to the tumor cells. Tumorsubstructures expected to be negative (stroma, vessels, and lymphocytes)did not exhibit positive staining with 353.2-1 or 353.9-20. Additionalstaining of normal tissues with 353-2.1 (FOLR1-2.1) are summarized inTable 13, below and shown in FIG. 14. Taken together, the IHCcharacterization data suggests that 353.2-1 and 353.9-20 are specific toFOLR1 in FFPE tissues (see FIG. 1 and FIG. 2).

TABLE 12 Optimized Assay Conditions Step Action/Reagent (Vendor) TimeBake Temperature: 60° C. 30 Minutes Dewax Bond Dewax Solution (Leica)Fixed 100% Ethanol (Pharmco Aaper) Antigen Retrieval Bond EpitopeRetrieval 2 20 Minutes (ethylenediaminetetraacetic acid based pH 9.0solution) Endogenous Peroxidase Peroxide (Leica)  5 Minutes Block TestArticle IMGN353.2-1 at 1.5 μg/mL 15 Minutes IMGN353.9-20 at 6.0 μg/mLDetection Post Primary Regent (Leica)  8 Minutes Polymer (Leica)  8Minutes Mixed DAB (Leica) 10 Minutes Counterstain Hematoxylin (Leica)  5Minutes

TABLE 13 Optimized Assay Conditions Normal Tissue, Structure 2.1Staining Adrenal Gland + Breast lobules + Fallopian tube, surfaceepithelium + Kidney, tubules + Pancreas, ducts + Pituitary, pituitarycells + Salivary gland, intercalated ducts + Breast, connective tissue −Esophagus submucosa & muscularis − Eye, cornea − Kidney, glomeruli −Lung, interalveolar connective tissue − Liver, hepatocytes − Pancreas,acinar cells − Luna, epithelium −/+ Stomach, surface epithelium, pits −

Example 3. Characterization of the Selected Anti-FOLR1 Antibodies

As described above in Example 2, of the fourteen hybridoma clonesselected based on primary and confirmation FACS screening, ten primaryclones were analyzed by immunohistochemistry (IHC) analysis. Of the tenprimary clones (i.e., 352.1, 352.2, 353.1, 353.2, 353.3, 353.5, 353.9,353.15, 354.1, and 354.2), five were positive by IHC (i.e., 353.1,353.2, and 353.3, 353.5, and 353.9), and all five were derived from thesame fusion (fusion 353). Four of the five were successfully subcloned.One subclone of primary clone 353.2 was chosen and named 353.2-1(“2.1”). One subclone of primary clone 353.3 was chosen and named353.3-8 (“3.8”). One subclone of primary clone 353.5 was chosen andnamed 353.5-7 (“5.7”), and two subclones of primary clone 353.9 werechosen and named 353.9-20 (“9.20”) and 353.9-21 (“9.21”). Subclones 9.20and 9.21 were sequenced, and as expected, both subclones had the samesequence. In addition, two of the clones, 2.1 and 9.20 were depositedwith ATCC as PTA-120197 and PTA-120196, respectively, on Apr. 16, 2013.

Specificity of the Anti-FOLR1 Antibodies by Western Blot

Specificity of the generated antibodies was analyzed by Western blotwith a panel of cell lysates prepared from FOLR1-positive (Igrov-1,Ovcar-3, Caov-3, Wish, and Skov-3) and FOLR1-negative (BxPC3, Panc-1,and ASPC1) cell lines. For the assay, lysates were run in SDSpolyacrylamide gel electrophoresis and transferred to a nitrocellulosemembrane by the standard procedures. The membrane was incubated with theanti-FOLR1 antibodies of the invention, and the formed antigen-antibodycomplexes were detected with secondary anti-murine antibodies conjugatedwith horse-radish peroxidase (hrp) (FIG. 3). All tested anti-FOLR1antibodies recognized FOLR1 in cell lines with high levels of FOLR1expression (i.e., Igrov-1 and Wish). FOLR1 in low expressing cell linesOvcar-3. Caov-3 and Skov-3 was detected only by anti-FOLR1 clones 2.1and 9.21; clones 3.8 and 5.7 did not stain these cell lysates perhapsdue to insufficient sensitivity of the antibodies. No additionalnon-specific bands were detected in FOLR1-positive cell lines by theclones; no staining of FOLR1-negative cell lines was observed.

Binding of the Anti-FOLR1 Antibodies to Denatured and not DenaturedCells

The ability of the anti-FOLR1 antibodies to bind to denatured andnon-denatured (native confirmation) FOLR1 was assayed by indirect FACSwith FOLR1-positive cells KB and T47D. Cells were harvested by Versineand washed with phosphate buffered saline (PBS). Denatured cells wereprepared by incubation of the cells in PBS containing 10% formaldehydeat 4° C. overnight followed by washing with PBS and incubation at 95° C.for 30 min. Denatured and non-denatured cells were then incubated withanti-FOLR1 antibodies diluted in FACS buffer (RPMI-1640 mediumsupplemented with 2% normal goat serum) on ice for 2 hours. The cellswere centrifuged, washed with PBS and incubated for 40 min withFITC-conjugated goat anti-mouse IgG-antibody. The cells were centrifugedagain, washed with PBS and resuspended with 0.2 ml of PBS containing 1%formaldehyde. Cell-associated fluorescence was measured using aFACSCalibur flow cytometer with the HTS multiwell and analyzed usingCellQuest Pro (BD Biosciences. San Diego, US). As shown on FIG. 4, allanti-FOLR1 antibodies bound to both denatured and not denatured cells.

Affinity of the Anti-FOLR1 Antibodies by ELISA

The binding affinity of the anti-FOLR1 antibodies was examined by ELISAwhere recombinant humanFOLR1-murine Fc2a protein was used as theantigen. The recombinant protein was immobilized on microtiter plates,and the antibodies were added at a range of concentrations to theplates. The plates were incubated for two hours at room temperature,washed with PBS supplemented with 0.05% Tween-20, and incubated withhrp-labeled goat anti-murine secondary antibody for one hour at roomtemperature. The plates were washed with PBS/Tween-20 again, and boundhrp-conjugated antibody was detected by adding the hrp-substrate TMB(Bio-FX). Representative results are shown in FIG. 5. The anti-FOLR1antibodies had similar affinity to human FOLR1 at half-maximal effectiveconcentration (EC50) of 0.5 to 0.9 nM.

No Cross-Reactivity of the Anti-FOLR1 Antibodies with FOLR2 and FOLR3

FOLR1 is a member of Folate Receptor family. Cross-reactivity of theanti-FOLR1 antibodies with the other members of the family FOLR2 andFOLR3 was assayed by ELISA. Recombinant protein FOLR2-His or FOLR3-His(R&D Systems) was immobilized to Ni-NTA plates (QIAGEN) and theanti-FOLR1 antibodies were added to the plates and incubated for 2 hoursat room temperature. As positive controls for FOLR2 and FOLR3 ELISApolyclonal anti-FOLR2 and FOLR3 antibodies (R&D systems), respectively,were used. The formed antibody-antigen complexes were detected withhrp-labeled goat anti-murine secondary antibody. As shown in FIG. 6, theanti-FOLR1 antibodies of the invention did not bind to FOLR2 or FOLR3;only the control antibodies detected corresponding antigens.

Example 4. Antigen Epitope Characterization

Human FOLR1 has three potential sites for N-glycosylation at positions69, 161 and 201 (UniProt), and, as reported in literature, all threesites are glycosylated. To characterize the nature of the epitopesrecognized by the anti-FOLR1 antibodies described herein, bindingexperiments were performed with deglycosylated and non-treated receptor.Of the generated anti-FOLR1 clones, only clone 2.1 was used in the studybecause, based on the sequencing data, the clones are related and likelyto bind to the same epitope. In addition to clone 2.1, two otheranti-FOLR1 antibodies were included: huMov19 (WO 2011/106528) and cloneBN3.2 (Leica). In order to deglycosylate FOLR1, recombinant human FOLR1or lysates of FOLR1-positive KB or Igrov-1 cells were treated with amixture of deglycosylation enzymes (Enzymatic DeGlycoMX Kit, QA-bio)according to the Manufacturer's protocol. Then, samples of treated andnon-treated FOLR1 were used in ELISA and Western blot analysis. For theELISA, deglycosylated and non-treated FOLR1 were immobilized to ELISAplates (Immulon), and the anti-FOLR1 antibodies FRIHC2-1 (“2.1”) orhuMov19 were added. After 2 h incubation, antibody-antigen complexeswere detected with hrp-labeled goat anti-human (for huMov19) oranti-murine (for 2.1) secondary antibody (FIG. 7). For the Western blotanalysis, samples of deglycosylated and non-treated lysates or huFOLR1recombinant protein were separated by SDS polyacrylamide gelelectrophoresis and transferred to a nitrocellulose membrane by thestandard procedures. The membrane was incubated with the anti-FOLR1antibodies 2.1, huMov19, or BN3.2, and the antigen-antibody complexeswere detected with the appropriate secondary anti-murine or anti humanantibodies conjugated with horse-radish peroxidase (FIG. 8). As shown inFIGS. 7 and 8, binding of antibody 2.1 to deglycosylated vs. non-treatedFOLR1 was significantly reduced suggesting the antibody binds to aglycodependent epitope. In contrast, the other two anti-FOLR1antibodies, huMov19 and BN3.2, bind similarly to deglycosylated andnon-treated receptor indicating that (i) the FOLR1 protein was notdamaged during the deglycosylation procedure and (ii) huMov19 and BN3.2recognize protein epitopes of FOLR1.

Example 5. Cloning and Sequencing of the VL and VH Regions of theAnti-Human FOLR1 Antibodies

Total cellular RNA was prepared from 5×10⁶ cells of the FOLR1 hybridomasdescribed in Example 1 using an RNeasy kit (QIAgen) according to themanufacturer's protocol. cDNA for the eight subclones clones (2.1, 2.12,3.8, 3.9, 5.7, 5.10, 9.20, and 9.21) was subsequently synthesized fromtotal RNA using the SuperScript II cDNA synthesis kit (Invitrogen).

The PCR procedures for amplifying the antibody variable region cDNAsderived from hybridoma cells were based on methods described in Wang etal. ((2000) J Immunol Methods. 233:167-77) and Co et al. ((1992) JImmunol. 148:1149-54). The variable light chain (VL) and variable heavychain (VH) sequences were amplified by degenerate primers on the 5′endand either murine kappa or IgG1 constant region specific primersrespectively on the 3′ end. The PCR reactions were then run on a 1% lowmelt agarose gel, followed by the excision of the 300 to 400 bp ampliconbands that were subsequently purified using Zymo DNA mini columns. Thepurified amplicons were sent to Beckman Coulter Genomics for sequencingutilizing the same 5′ and 3′ primers of the PCR reactions in order togenerate the variable region cDNA sequences from both directions.

Since the degenerate primers used to clone the VL and VH cDNA sequencesalter the 5′ end, additional sequencing efforts were needed to verifythe complete cDNA sequences. The preliminary sequences were entered intoa search query of the NCBI IgBlast site (www.ncbi.nlm.nih.gov/igblast/)to identify the murine germline sequences from which the antibodysequences had been derived. PCR primers were then designed to anneal tothe germline linked leader sequence of the murine antibody so that thisnew PCR reaction would yield a complete variable region cDNA sequence,unaltered by the PCR primers. The PCR reactions, band purifications, andsequencing were performed as described above.

Mass Determination for Sequence Confirmation

The variable regions cDNA sequences obtained for each of the anti-FOLR1antibodies were combined with germline constant region sequences toobtain full length antibody cDNA sequences. The molecular weights of theheavy and light chains were then calculated from translations of thecDNA sequences and compared with the molecular weights obtained by LC/MSanalyses of the purified murine anti-FOLR1 antibodies. The LC/MS is doneby deglycosylating and reducing the antibody to isolate full chain lightand heavy chain peptides. The observed molecular weights for each of theheavy chains matched the expected, but each of the light chains was offby approximately 85 Da. Subsequent peptide fragmentation analysis byLC/MS of the light chain fragments indicated that the final serine oflight chain leader peptide was in fact retained on the mature lightchain, adding about 87 Da to the expected MW, thus confirming the cDNAsequences for each of the FOLR1 antibodies.

Composite CDR Sequences for the Anti-FOLR1 Antibodies

Alignments of the antibody sequences for the 8 subclones revealed that 3of the 4 original hybridomas had produced closely related, but unique,antibodies. As expected, each of the 4 sister subclone pairs wereidentical. In addition two sets of subclones were also identicalresulting in the 3 unique antibody sequences (SEQ ID NOs:27-32) (2.1,5.7, and 9.20). The light and heavy chain variable framework sequencesof these 3 unique antibodies are closely related, but each antibodycontains unique CDRs, likely as a result of somatic amino acidsubstitutions (see Table 14 below). Because these CDR variants of themurine anti-FOLR1 antibodies were found to be functionally identical,they provide some structural insight into the sequence flexibility ofthe CDRs of the anti-FOLR1 antibodies of the invention. Light chain CDRs2 and 3 were identical in each of the antibodies suggesting that thesetightly conserved CDRs can provide a consistent structural basis forFOLR1 binding. On the other hand, the amino acid substitutions in theremaining CDRs, particularly those in heavy chain CDRs 2 and 3, suggestthat these positions are critical for refinement of the affinity andspecificity of these antibodies. The specific residue substitutions inthese CDR positions also provide examples of residues that can beincorporated within engineered versions of these antibodies. Table 14provides a composite CDR sequence listing compiled from the anti-FOLR1antibodies of the invention. The composite CDRs identified herein can beused for the design of recombinant antibodies that would be expected topreserve the functional attributes of the anti-FOLR1 antibodies of thepresent invention.

TABLE 14 Composite CDRS Anti-FOLR1 composite CDRs Light ChainCDR1: KS[T/S][K/E]SLLNSDGFTYLD (SEQ ID NO: 24)CDR2: LVSNHFS (SEQ ID NO: 25) CDR3: FQSNYLPLT (SEQ ID NO: 26)Heavy Chain CDR1: N[Y/S]YIH (SEQ ID NO: 21)CDR2: WIYP[G/E][S/N][F/V/L]N[V/T][E/R/Q]YN[E/D] KFKA (SEQ ID NO: 22)CDR3: RGIY[F/Y]YSPYA[L/M]D[Y/H] (SEQ ID NO: 23)

Antibody Humanization

The FRIHC2-1 antibody was humanized following resurfacing methodspreviously described, such as, for example in Roguska et al., Proc.Natl. Acad. Sci., USA, 91(3):969-973 (1994) and Roguska et al., ProteinEng. 9(10):895-904 (1996), which are incorporated in their entiretyherein by reference. Resurfacing involves identification of the variableregion framework surface residues in both the light and heavy chains andreplacing them with human equivalents. The murine CDRs are preserved inthe resurfaced antibody. Exemplary CDRs of FRIHC2-1 antibody are definedas indicated in Table 14. To minimize concerns about the impact ofconjugating lysines that fall in CDRs, lysine 24 and lysine 27 in murineFRIHC2-1 antibody light chain CDR1 were replaced with arginine forhumanized version 1.0 (shown in italic), so both versions of the LC CDR1are given. In addition to the AbM heavy chain CDR2 definition employedfor resurfacing, the table provides exemplary Kabat defined heavy chainCDR2s for both the murine and human versions of FRIHC2-1 antibody. Theunderlined sequence marks the portion of the Kabat heavy chain CDR2 thatwas not considered a CDR for resurfacing.

Surface residue positions were defined as any position with a relativeaccessibility of 30% or greater (Pedersen J. T. et. Al, J. Mol. Biol.1994, 235: 959-973). The calculated surface residues were then alignedwith human germline surface sequences to identify the most homologoushuman surface sequence. The human germline sequence used as thereplacement surface for the VL domains of FRIHC2-1 antibody wasIGKV2-30*01 while IHV1-69*10 was used as the replacement surface forFRIHC2-1 antibody VH. The specific framework surface residue changes forFRIHC2-1 antibody are given in FIG. 9. Since the resurfaced light chainincluded the CDR1 lysine substitutions in the preferred version, aresurfaced version (v1.01) was also generated with murine lysinesretained in CDR-L1. FIG. 10 shows the alignment of the resurfacedsequences for FRIHC2-1 variable domain of both light and heavy chainwith their murine counterparts.

In addition to humanization by variable domain resurfacing, FRIHC2-1antibody was also humanized following complementary determining region(CDR) grafting technology (Jones et al., Nature 321: 604-608 (1986) andVerhoeyen et al., Science 239: 1534-1536 (1988)). The CDR graftingmethod consists of grafting the CDRs from a naturally evolved murineantibody onto the Fv framework regions (FRs) of a human antibody. TheKabat numbering scheme and Kabat CDR definitions were used for CDRgrafting of the FRIHC2-1 antibody. Exemplary CDRs of FRIHC2-1 for CDRgrafting are given in Table 14. The human immunoglobulin germlinesequence with the highest homology to the murine FRIHC2-1 antibody wasidentified through the interactive tool, V-QUEST, of the InternationalImMunoGeneTics information System® (IMGT (http://imgt.cines.fr/) asdescribed in Lefranc, Nucleic Acids Res. 29: 207-209 (2001). The humangermline sequences used as the acceptor frameworks for the VL and VHdomains of FRIHC2-1 antibody were IGKV2D-29*02 and IGHV1-2*02,respectively. To minimize concerns about the impact of conjugatinglysines that fall in CDRs, lysine 24 and lysine 27 in murine FRIHC2-1antibody light chain CDR1 were replaced with arginine in the CDR graftedconstructs (Table 15). The specific framework residue changes as well asthe substitution in CDR-L1 in CDR-grafting of FRIHC2-1 antibody aregiven in FIG. 11, and the alignments of the CDR-grafted sequences forthe FRIHC2-1 antibody variable domains with its murine counterparts areillustrated in FIG. 12.

TABLE 15 FRIHC2-1 CDR's Resurfacing FRIHC2-1 CDR's (CDR grafting)Light Chain Light Chain Murine and resurfaced v1.01 CDR1:Murine and CDR-grafted v1.01 CDR1: KSSKSLLNSDCIFTYLD (SEQ ID NO: 6)KSSKSLLNSDGFTYLD (SEQ ID NO: 6) Resurfaced v1.0 CDR1:CDR-grafted v1.0 CDR1: RSSRSLLNSDGFTYLD (SEQ ID NO: 59)RSSRSLLNSDGFTYLD (SEQ ID NO: 59) CDR2: LVSNHFS (SEQ ID NO: 7)CDR2: LVSNHFS (SEQ ID NO: 7) CDR3: FQSNYLPLT (SEQ ID NO: 8)CDR3: FQSNYLPLT (SEQ ID NO: 8) Heavy Chain Heavy ChainCDR1: NSYIH (SEQ ID NO: 3) CDR1: NSYIH (SEQ ID NO: 3)CDR2: WIYPESLNTQ (SEQ ID NO: 60) CDR2: WIYPESLNTQYNEKFKA (SEQ ID NO: 4)CDR3: RGIYYYSPYALDH (SEQ ID CDR3: RGIYYYSPYALDH (SEQ ID NO: 5) NO: 5)Kabat FRHIC2-1 HC CDR2 Murine HC CDR2: WIYPESLNTQYNEKFKA (SEQ ID NO: 4)Resurfaced HC CDR2: WIYPESLNTQYNQKFQG (SEQ ID NO: 61)

Example 6. IHC Evaluation of 353-2.1 (FOLR1-2.1) Antibody Using HumanTumor Samples

Human tumor samples representative of ovarian cancer (n=63), lungadenocarcinoma (n=104), and endometrial adenocarcinoma (n=58) wereevaluated for FOLR1 expression by IHC using the 353-2.1 antibody. Theintensity of FOLR1 staining and the distribution of scores aresummarized in Table 16, below. FIG. 15 shows an example of staining ofovarian cancer and lung adenocarcinoma tissue with the 353-2.1 antibody.These results demonstrate the utility of 353-2.1 as a specific andsensitive antibody for use in IHC assays to identify patients aspotential candidates for therapy with FOLR1 targeting agents (e.g.,IMGN853).

TABLE 16 Distribution of Scores (% Positivity) LUNG ENDOMETRIAL OVARIANADENO- ADENO- CANCER CARCINOMA CARCINOMA TUMOR TYPE: n = 63 n = 1.04 n =58 Positive (any 65% 70% 64% intensity): ≥level 2 intensity 59% 47% 33%with at least 25% tumor cells stained: ≥level 3 intensity 51% 19% 14%with at least 25% tumor cells stained:

The unique antigen specificity and high binding affinity of theFOLR1-2.1 (FOLR1 353-2.1) antibody was further demonstrated using anadditional IHC assay. This IHC assay utilizes the OptiView DAB DetectionKit on a Ventana BenchMark XT automated slide stainer forsemi-quantitative determination of FOLR1 protein expression informalin-fixed paraffin embedded tissue samples. The assay has beenoptimized and validated with respect to specificity, sensitivity, andprecision using normal and tumor tissue controls. Under the optimizedcondition, sharp membranous staining was clearly observed in tumor cellswhereas normal stromal tissues were completely negative. (FIG. 16). Inaddition, this assay also achieved a broader dynamic range therebyallowing better discrimination of moderate staining intensity (level 2,medium brown staining, FIG. 17) from the strongest staining intensity(level 3, dark brown staining, FIG. 17). The enhanced dynamic rangeimproves the ability to rank FOLR1 positive samples based on stainingintensity and enable further identification a sub-popupation of patientswith the highest level of FOLR1 expression.

A BN3.2 (Leica) antibody and the FOLR1-2.1 (FOLR1 353-2.1) antibody werecompared using an ovarian cancer tissue micro array (TMA). Using theBN3.2 (Leica) antibody in an IHC assay (BN3.2 assay), close to 50% ofsamples (16 out of 35) were scored in the highest category (level 3staining intensity on at least 25% tumor cells). In contrast, using theFOLR1-2.1 (FOLR1 353-2.1) antibody in the IHC assay described aboveutilizing the OptiView DAB Detection Kit on a Ventana BenchMark XTautomated slide stainer (FOLR1-2.1 assay), allowed futher separation ofthese 16 samples into 2 different categories: 6 in the highest category(level 3 staining intensity on at least 25% tumor cells, Table 17), andthe other 10 in the second highest category (level 2 staining intensityon at least 25% tumor cells, Table 17). Thus, the more discreet stainingobtained with the FOLR1-2.1 antibody in the FOLR1-2.1 assay allows fordiscrimination among samples all grouped together as samples with level3 expression using the BN3.2 antibody in the BN3.2 assay.

TABLE 17 FOLR1 prevalence comparison in ovarian cancer TMA (n = 35)Score FOLR1-2.1 assay BN3.2 assay Positive (any intensity) 24 (69%) 28(80%) ≥level 1 intensity with at 21 (60%) 27 (77%) least 25% tumor cellsstained: ≥level 2 intensity with at 17 (49%) 25 (71%) least 25% tumorcells stained: ≥level 3 intensity with at  6 (17%) 16 (46%) least 25%tumor cells stained:

All publications, patents, patent applications, internet sites, andaccession numbers/database sequences (including both polynucleotide andpolypeptide sequences) cited herein are hereby incorporated by referencein their entirety for all purposes to the same extent as if eachindividual publication, patent, patent application, internet site, oraccession number/database sequence were specifically and individuallyindicated to be so incorporated by reference.

1-24. (canceled)
 25. A method of detecting FOLR1 expression in a samplecomprising contacting said sample with an antibody, antigen-bindingfragment thereof, or polypeptide that specifically binds to the sameFOLR1 epitope as an antibody selected from the group consisting of: (a)an antibody comprising the polypeptide of SEQ ID NO:27 and thepolypeptide of SEQ ID NO:28; (b) an antibody comprising the polypeptideof SEQ ID NO:29 and the polypeptide of SEQ ID NO:30; (c) an antibodycomprising the polypeptide of SEQ ID NO:31 and the polypeptide of SEQ IDNO:32; (d) an antibody comprising the polypeptide of SEQ ID NO:62 andthe polypeptide of SEQ ID NO:63 or SEQ ID NO:64; and (e) an antibodycomprising the polypeptide of SEQ ID NO:65 and the polypeptide of SEQ IDNO:66 or SEQ ID NO:67. 26-29. (canceled)
 30. The method of claim 90,wherein the detecting is by IHC. 31-46. (canceled)
 47. The method ofclaim 90, wherein the detecting is by enzyme linked immunosorbent assay(ELISA). 48-89. (canceled)
 90. A method of detecting FOLR1 expression ina sample comprising contacting said sample with an antibody,antigen-binding fragment thereof, or polypeptide comprises the VH CDR1-3and VL CDR1-3 polypeptide sequences selected from the group consistingof: (a) SEQ ID NOs:3-8, respectively; (b) SEQ ID NOs:9-14, respectively;(c) SEQ ID NOs: 15-20, respectively; (d) SEQ ID NOs:21-26, respectively;(e) SEQ ID NOs: 3-5 and SEQ ID NOs: 59, 7, and 8, respectively; (f) SEQID NOs: 3, 60, and 5 and SEQ ID NOs: 6-8, respectively; (g) SEQ ID NOs:3, 61, and 5 and SEQ ID NOs: 6-8, respectively; (h) SEQ ID NOs: 3, 60,and 5 and SEQ ID NOs: 59, 7, and 8, respectively; and (i) SEQ ID NOs: 3,61, and 5 and SEQ ID NOs: 59, 7, and 8, respectively.
 91. The method ofclaim 90, wherein the antibody, antigen-binding fragment thereof, orpolypeptide comprises: (a) a heavy chain variable region (VH) comprisingthe amino acid sequence of the polypeptide of SEQ ID NO:27 and a lightchain variable region (VL) comprising the amino acid sequence thepolypeptide of SEQ ID NO:28; (b) a heavy chain variable region (VH)comprising the amino acid sequence of the polypeptide of SEQ ID NO:29and a light chain variable region (VL) comprising the amino acidsequence the polypeptide of SEQ ID NO:30; (c) a heavy chain variableregion (VH) comprising the amino acid sequence of the polypeptide of SEQID NO:31 and a light chain variable region (VL) comprising the aminoacid sequence the polypeptide of SEQ ID NO:32; (d) a heavy chainvariable region (VH) comprising the amino acid sequence of thepolypeptide of SEQ ID NO:62 and a light chain variable region (VL)comprising the amino acid sequence the polypeptide of SEQ ID NO:63 orSEQ ID NO:64; or (e) a heavy chain variable region (VH) comprising theamino acid sequence of the polypeptide of SEQ ID NO:65 and a light chainvariable region (VL) comprising the amino acid sequence the polypeptideof SEQ ID NO:66 or SEQ ID NO:67.
 92. The method of claim 90, wherein theantibody, antigen-binding fragment thereof, or polypeptide comprises:(a) a heavy chain comprising the amino acid sequence of SEQ ID NO:33 anda light chain comprising the amino acid sequence of SEQ ID NO:34; (b) aheavy chain comprising the amino acid sequence of SEQ ID NO:35 and alight chain comprising the amino acid sequence of SEQ ID NO:36; or (c) aheavy chain comprising the amino acid sequence of SEQ ID NO:37 and alight chain comprising the amino acid sequence of SEQ ID NO:38.
 93. Themethod of claim 30, wherein the sample is a cancer sample.
 94. Themethod of claim 93, wherein a FOLR1 score of at least 2 in 25% orgreater of the cells identifies the cancer as likely to respond to anactive agent comprising an anti-FOLR1 antibody or antigen-bindingfragment thereof or indicates that the patient will benefit fromadministration of an active agent comprising an anti-FOLR1 antibody orantigen-binding fragment thereof.
 95. The method of claim 93, wherein aFOLR1 score of at least 2 in 25% to 75% of the cells identifies thecancer as likely to respond to an active agent comprising an anti-FOLR1antibody or antigen-binding fragment thereof or indicates that thepatient will benefit from administration of an active agent comprisingan anti-FOLR1 antibody or antigen-binding fragment thereof.
 96. Themethod of claim 93, wherein a FOLR1 score of at least 2 in greater than75% of the cells identifies the cancer as likely to respond to an activeagent comprising an anti-FOLR1 antibody or antigen-binding fragmentthereof or indicates that the patient will benefit from administrationof an active agent comprising an anti-FOLR1 antibody or antigen-bindingfragment thereof.
 97. The method of claim 93, wherein a FOLR1 score ofat least 3 in 25% or greater of the cells identifies the cancer aslikely to respond to an active agent comprising an anti-FOLR1 antibodyor antigen-binding fragment thereof or indicates that the patient willbenefit from administration of an active agent comprising an anti-FOLR1antibody or antigen-binding fragment thereof.
 98. The method of claim93, wherein a FOLR1 score of at least 3 in 25% to 75% of the cellsidentifies the cancer as likely to respond to an active agent comprisingan anti-FOLR1 antibody or antigen-binding fragment thereof or indicatesthat the patient will benefit from administration of an active agentcomprising an anti-FOLR1 antibody or antigen-binding fragment thereof.99. The method of claim 93, wherein a FOLR1 score of at least 3 ingreater than 75% of the cells identifies the cancer as likely to respondto an active agent comprising an anti-FOLR1 antibody or antigen-bindingfragment thereof or indicates that the patient will benefit fromadministration of an active agent comprising an anti-FOLR1 antibody orantigen-binding fragment thereof.
 100. The method of claim 93, whereinthe cancer sample is a bodily fluid, cell, or tissue sample.
 101. Themethod of claim 100, wherein said bodily fluid is blood, ascites, urine,plasma, serum, or peripheral blood.
 102. The method of claim 93, whereinsaid cancer is selected from the group consisting of cervical cancer,ovarian cancer, brain cancer, breast cancer, uterine cancer, endometrialcancer, pancreatic cancer, renal cancer, lung cancer, and cancer of theperitoneum.
 103. The method of claim 102, wherein the ovarian cancer isepithelial ovarian cancer.
 104. The method of claim 102, wherein thecancer is platinum resistant, relapsed, or refractory.
 105. The methodof claim 90, wherein the antibody or antigen-binding fragment thereof ischimeric or humanized.
 106. The method of claim 90, wherein the antibodyor antigen-binding fragment thereof is a full-length antibody.
 107. Themethod of claim 90, wherein the antibody or antigen-binding fragmentthereof is an antigen-binding fragment.
 108. The method of claim 90,wherein the antibody or antigen-binding fragment thereof binds to ahuman folate receptor 1 with a Kd of about 0.5 to about 10 nM.
 109. Themethod of claim 90, wherein the antibody, antigen-binding fragmentthereof, or polypeptide further comprises a detectable label.
 110. Themethod of claim 109, wherein said detectable label is selected from thegroup consisting of immunofluorescent label, chemiluminescent label,phosphorescent label, enzyme label, radiolabel, luminophore,avidin/biotin, colloidal gold particles, colored particles, and magneticparticles.
 111. The method of claim 109, wherein said detectable labelis selected from the group consisting of biotin, digoxigenin,fluorescein, tritium, and rhodamine.